Telomerase activating compounds and methods of use thereof

ABSTRACT

The present invention is directed to use of a series of compounds and compositions comprising the same for activating telomerase and treating diseases, disorders and/or conditions related thereto.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.12/602,956, filed Jun. 17, 2010, which in turn is a 371 of PCTInternational Application No. PCT/IL2008/000756, filed Jun. 4, 2008,which claims the benefit of U.S. Provisional Application Nos.60/924,875, filed Jun. 4, 2007, 60/929,524, filed Jul. 2, 2007,60/929,525, filed Jul. 2, 2007 and 61/006,924, filed Feb. 6, 2008. Thedisclosures of all applications are herein incorporated by reference.

FIELD OF THE INVENTION

The present invention is directed to use of a series of compounds andcompositions comprising the same for enhancing expression and/oractivating telomerase and for treating diseases, disorders and/orconditions related thereto.

BACKGROUND OF THE INVENTION

Telomerase is a ribonucleoprotein that catalyzes the addition oftelomeric repeats to the ends of telomeres. Telomeres are long stretchesof repeated sequences that cap the ends of chromosomes. In humans,telomeres are typically 7-10 kb in length and comprise multiple repeats.Telomerase is not expressed in most adult cells, and telomere lengthdecreases with successive rounds of replication

Telomerase acts as reverse transcriptase in the elongation of telomeres,which prevent the loss of telomeres due to the end replication problems.Without telomerase the telomeres are shortened at each cell divisionwhich leads to senescence, apoptosis and cell death caused by chromosomeinstability. Telomerase is inactive in somatic cells but active in 90%of cancer cells, where telomerase is reactivated. Although telomeraseactivation may be dangerous, because it can mimic the cancer developmentprocess, telomerase enhancing agents may be theoretically applicable asanti-aging agents and clinically useful in certain medical conditions.In contrast, telomerase inhibitors may be useful to fight cancer. Cancerand aging are closely inter-related: Interventions that protect againstcancer can lead to premature aging while immortalization of cells isrequired in the formation of malignant cancer cells. Despite thetheoretical risk of activation of carcinogenesis, activation oftelomerase may lead to reduced rate of aging.

The assessment of the telomere length is important in the understandingof biological and clinical significance of the telomere. The telomerelength serves as a useful indicator in the study of the chromosomalstability, telomerase activity and/or expression, proliferative capacityand aging process of the cells. The clinical value of telomeres can bedemonstrated in its importance in cancer, premature aging syndrome orsegmental progeria; genetic anomalies, diseases resulting fromchromosomal instability, such as Bloom syndrome (a rare inheriteddisorder characterized by a high frequency of breaks and rearrangementsin an affected person's chromosomes), and age-related diseases, such asWarner's Syndrome (a rare illness that manifests rapid aging in youngerpeople). The dynamics of telomere length have distinct patterns ofexpression in specific disease progressions. Therefore it has a greatvalue in the prognosis of the diseases.

Telomere length can be measured by southern blot, hybridizationprotection assay, fluorescence in situ hybridization, flow cytometry,primed in situ, quantitative-polymerase chain reaction and singletelomere length analysis.

Lack of telomerase activity and/or expression and short telomeres maycause dyskeratosis congenita, aplastic anemia, increase of death due tocardiovascular diseases, strokes or infections, hypertension or chronicstress.

It was shown that transduction of telomerase in telomerase knockout miceprevented damage in the liver.

In addition to the role of telomerase in telomere length maintenance,accumulating data suggest that the telomerase reverse transcriptase(TERT) protein has additional physiological functions, i.e. protectingcells and mice from various damages in a mechanism (yet unclear) thatdoes not involve telomere elongation.

Compounds which activate telomerase will thus find application inmultiple clinically relevant scenarios.

SUMMARY OF THE INVENTION

In one embodiment, this invention provides a method of stimulating orincreasing telomerase activity and/or expression in a cell or tissue,comprising contacting said cell or tissue with a compound represented bythe structure of formula I:

wherein

Z is carbon, nitrogen, phosphor, arsenic, silicon or germanium;

R₁ to R₉ are the same or different, H, D, OH, halogen, nitro, CN,nitrileamido, amidosulfide, amino, aldehyde, substituted ketone, —COOH,ester, trifluoromethyl, amide, substituted or unsubstituted alkyl,alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl,arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl, alkylhaloalkyl,haloaryl, aryloxy, amino, monoalkylamino, dialkylamino, alkylamido,arylamino, arylamido, alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or R₃, R₄, or R₇, forms a fusedcycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with themain aromatic ring; and

R₁₀ is nothing, H, D, OH, halogen, oxo, nitro, CN, nitrileamido,amidosulfide, amino, aldehyde, substituted ketone, —COOH, ester,trifluoromethyl, amide, substituted or unsubstituted alkyl, alkenyl,alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl,alkoxy, haloalkyl, haloaryl, cycloalkyl, alkylcycloalkyl, aryloxy,monoalkylamino, dialkylamino, alkylamido, arylamino, arylamido,alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl,heterocycloalkylalkyl, heteroaryl, hetroarylalkyl, alkylheteroaryl; orits isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof.

In one embodiment, this invention provides a method of treating acondition capable of being affected by telomerase activation and/orexpression in a subject, comprising administering to said subject acompound represented by the structure of formula I:

wherein

Z is carbon, nitrogen, phosphor, arsenic, silicon or germanium;

R₁ to R₉ are the same or different, H, D, OH, halogen, nitro, CN,nitrileamido, amidosulfide, amino, aldehyde, substituted ketone, —COOH,ester, trifluoromethyl, amide, substituted or unsubstituted alkyl,alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl,arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl, alkylhaloalkyl,haloaryl, aryloxy, amino, monoalkylamino, dialkylamino, alkylamido,arylamino, arylamido, alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or R₃, R₄, or R₇, forms a fusedcycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with themain aromatic ring; and

R₁₀ is nothing, H, D, OH, halogen, oxo, nitro, CN, nitrileamido,amidosulfide, amino, aldehyde, substituted ketone, —COOH, ester,trifluoromethyl, amide, substituted or unsubstituted alkyl, alkenyl,alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl,alkoxy, haloalkyl, haloaryl, cycloalkyl, alkylcycloalkyl, aryloxy,monoalkylamino, dialkylamino, alkylamido, arylamino, arylamido,alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl,heterocycloalkylalkyl, heteroaryl, hetroarylalkyl, alkylheteroaryl; orits isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof; whereby saidcompound stimulates or enhances telomerase activity and/or expression.

In another embodiment, said condition is HIV infection or a degenerativedisease. In another embodiment, said degenerative disease isneurodegenerative disease, a degenerative disease of the bones orjoints, macular degeneration, atherosclerosis, or anemia. In oneembodiment, said condition is an inflammatory disease. In anotherembodiment, said condition is dyskeratosis congenita. In anotherembodiment, the condition is aplastic anemia. In another embodiment,said condition is cancer.

In one embodiment, this invention provides a method of treating an acuteor chronic condition of the skin, comprising contacting skin with acompound represented by the structure of formula I:

wherein

Z is carbon, nitrogen, phosphor, arsenic, silicon or germanium;

R₁ to R₉ are the same or different, H, D, OH, halogen, nitro, CN,nitrileamido, amidosulfide, amino, aldehyde, substituted ketone, —COOH,ester, trifluoromethyl, amide, substituted or unsubstituted alkyl,alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl,arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl, alkylhaloalkyl,haloaryl, aryloxy, amino, monoalkylamino, dialkylamino, alkylamido,arylamino, arylamido, alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or R₃, R₄, or R₇, forms a fusedcycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with themain aromatic ring; and

R₁₀ is nothing, H, D, OH, halogen, oxo, nitro, CN, nitrileamido,amidosulfide, amino, aldehyde, substituted ketone, —COOH, ester,trifluoromethyl, amide, substituted or unsubstituted alkyl, alkenyl,alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl,alkoxy, haloalkyl, haloaryl, cycloalkyl, alkylcycloalkyl, aryloxy,monoalkylamino, dialkylamino, alkylamido, arylamino, arylamido,alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl,heterocycloalkylalkyl, heteroaryl, hetroarylalkyl, alkylheteroaryl; orits isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof.

In another embodiment, said acute or chronic condition is a wound, aburn, an abrasion, an incision, a graft site, a vascular lesion, alesion caused by an infectious agent, a chronic venous ulcer, a diabeticulcer, a compression ulcer, a pressure sore, a mucosal sore or ulcer,and keloid formation.

In one embodiment, this invention provides a method of inhibiting,abrogating or delaying cellular senescence in a subject, comprisingadministering to said subject, a compound represented by the structureof formula I:

wherein

Z is carbon, nitrogen, phosphor, arsenic, silicon or germanium;

R₁ to R₉ are the same or different, H, D, OH, halogen, nitro, CN,nitrileamido, amidosulfide, amino, aldehyde, substituted ketone, —COOH,ester, trifluoromethyl, amide, substituted or unsubstituted alkyl,alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl,arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl, alkylhaloalkyl,haloaryl, aryloxy, amino, monoalkylamino, dialkylamino, alkylamido,arylamino, arylamido, alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or R₃, R₄, or R₇, forms a fusedcycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with themain aromatic ring; and

R₁₀ is nothing, H, D, OH, halogen, oxo, nitro, CN, nitrileamido,amidosulfide, amino, aldehyde, substituted ketone, —COOH, ester,trifluoromethyl, amide, substituted or unsubstituted alkyl, alkenyl,alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl,alkoxy, haloalkyl, haloaryl, cycloalkyl, alkylcycloalkyl, aryloxy,monoalkylamino, dialkylamino, alkylamido, arylamino, arylamido,alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl,heterocycloalkylalkyl, heteroaryl, hetroarylalkyl, alkylheteroaryl; orits isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof.

In another embodiment, the method of inhibiting, abrogating or delayingcellular senescence in said subject extends a lifespan in said subject.

In one embodiment, this invention provides a method of diminishing orabrogating the effects of aging in a subject, comprising administeringto said subject a compound represented by the structure of formula I:

wherein

Z is carbon, nitrogen, phosphor, arsenic, silicon or germanium;

R₁ to R₉ are the same or different, H, D, OH, halogen, nitro, CN,nitrileamido, amidosulfide, amino, aldehyde, substituted ketone, —COOH,ester, trifluoromethyl, amide, substituted or unsubstituted alkyl,alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl,arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl, alkylhaloalkyl,haloaryl, aryloxy, amino, monoalkylamino, dialkylamino, alkylamido,arylamino, arylamido, alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or R₃, R₄, or R₇, forms a fusedcycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with themain aromatic ring; and

R₁₀ is nothing, H, D, OH, halogen, oxo, nitro, CN, nitrileamido,amidosulfide, amino, aldehyde, substituted ketone, —COOH, ester,trifluoromethyl, amide, substituted or unsubstituted alkyl, alkenyl,alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl,alkoxy, haloalkyl, haloaryl, cycloalkyl, alkylcycloalkyl, aryloxy,monoalkylamino, dialkylamino, alkylamido, arylamino, arylamido,alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl,heterocycloalkylalkyl, heteroaryl, hetroarylalkyl, alkylheteroaryl; orits isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof.

In another embodiment, the effects of aging comprise effects of theskin, eyes, musculature, or bones of the subject.

In another embodiment the structure of formula I is represented by thestructure of formula IV:

wherein R₁, R₃, R₄, R₆, R₇, R₉ and R₁₀ are as described above.

In another embodiment the structure of formula I is represented by thestructure of formula VI:

wherein R₁′, R₃′, R₄′, R₆′ R₇′, and R₉′ are the same or differentcomprising halogen, aryl, alkyl, cycloalkyl, heterocycloalkyl, alkoxy,monoalkylamino, dialkylamino or arylamino; and

R₁₀ is as described above.

In another embodiment the structure of formula I is represented by thestructure of formula VII:

In another embodiment the structure of formula I is represented by thestructure of formula VIII:

In another embodiment the structure of formula I is represented by thestructure of formula IX:

In another embodiment the structure of formula I is represented by thestructure of formula X:

In another embodiment the structure of formula I is represented by thestructure of formula XI:

In another embodiment the structure of formula I is represented by thestructure of formula XII:

In another embodiment the structure of formula I is represented by thestructure of formula XIII:

In another embodiment the structure of formula I is represented by thestructure of formula XIV:

In another embodiment the structure of formula I is represented by thestructure of formula XV:

In another embodiment the structure of formula I is represented by thestructure of formula XVI:

In another embodiment, the invention makes use of pharmaceuticalcompositions comprising compounds as described herein for any method asdescribed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The subject matter regarded as the invention is particularly pointed outand distinctly claimed in the concluding portion of the specification.The invention, however, both as to organization and method of operation,together with objects, features, and advantages thereof, may best beunderstood by reference to the following detailed description when readwith the accompanying drawings in which:

FIG. 1: Treatment of U-251 cell with compounds of the present inventionand measurement of telomerase activity by TRAP assay. A. Telomeraseactivity in cells treated with Compound 68. B. Quantification of resultsand % telomerase activation.

FIG. 2: Time-dependent increase of telomerase protein level by compoundsof the present invention. A. Western blot analysis with anti-humantelomerase antibody. B. Quantification of telomerase protein level.

FIG. 3: Compound 68 increases the expression of telomerase RNA. A.Northern blot analysis using hTERT-specific cDNA probe. AD isactinomycin D. B. Quantification of RNA level.

FIG. 4: The effect of treatment with compounds of the present inventionon survival and proliferation of hMSC.

FIG. 5: Activation of telomerase expression in hMSC by compounds of thepresent invention. A.—hMSC were treated with 250 nM of Compounds 79, 77and 68 for 6 hours. Immunofluorescence was performed with anti-hTERTantibody (red) and the nucleus was stained with DAPI (blue). B. hMSCtreated with compound 79 for 6 and 24 h. Telomerase activity wasmeasured by real time PCR using a quantitative telomerase detection kit(Allied Biotech Inc., USA).

FIG. 6: The effect of compounds of the present invention on senescenthuman keratinocytes in vitro.

FIG. 7: A. The effect of compounds of the present invention on humanretinal pigmented epithelial cells under oxidative stress. B. The effectof compounds on telomerase activity in RPE cells (measured by aquantitative telomerase detection kit-real time PCR).

FIG. 8: Lifespan extension of C. elegans by telomerase-activatingCompounds 68 and 77.

FIG. 9: Activation of telomerase expression by compounds of the presentinvention in rat endometrial cells. A,B. Histological analysis. C-F.Immunohistochemical analysis with specific anti-hTERT antibody. G.Telomerase activity in nuclear extracts derived from endometrium of ratsinjected with compounds of the present invention. G(B) Telomeraseactivity in nuclear extracts derived from endometrium of rats injectedwith compounds of the present invention measured by real time PCR usinga quantitative telomerase detection kit (Allied Biotech Inc., USA).

FIG. 10: Telomerase activation of rat brain cortex cells by compounds ofthe present invention.

FIG. 11: Telomerase activation of mouse CNS by compounds of the presentinvention.

FIG. 12: Telomerase protein is increased in rats treated with compoundsof the present invention.

FIG. 13: Prevention of glutamate-induced apoptosis in mouse cerebellumby Compound 79.

FIG. 14: Telomerase expression is activated in mouse heart by Compound79.

FIG. 15: Compound 68 prevents the effect of damaging drugs on embryodevelopment in rats.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

In the following detailed description, numerous specific details are setforth in order to provide a thorough understanding of the invention.However, it will be understood by those skilled in the art that thepresent invention may be practiced without these specific details. Inother instances, well-known methods, procedures, and components have notbeen described in detail so as not to obscure the present invention.

The present invention relates, in some embodiments, to the use of anovel class of tri-phenyl compounds and compositions comprising the samefor the treatment of, inter alia, diseases or conditions capable ofbeing affected by enhanced telomerase expression and/or telomeraseactivation.

The invention makes use of such compounds which stimulate and/orincrease telomerase expression and/or activity in the cells and tissuesof a subject, where the activity is decreased, missing, altered ornormal. Such disorders include, inter alia, a) Alzheimer's disease; b)Parkinson's disease; c) Huntington's disease; d) nerve damage, motorneuron disease, multiple sclerosis (MS), peripheral and central nervoussystem injury including spinal injury and cerebral vascular incidents;e) stroke; f) diseases or conditions associated with aging, such as forexample, aging of the skin such as dermal atrophy and thinning,elastolysis and skin wrinkling, sebaceous gland hyperplasia orhypoplasia, senile lentigo, pigmentation abnormalities, graying of hairand hair loss or thinning (baldness, alopecia), or chronic skin ulcers;g) degenerative joint disease; h) osteoporosis, osteoarthritis and otherdegenerative conditions of the skeletal system; i) sarcopenia and otherdegenerative conditions of the musculature; j) age- and stress-relateddiseases of the vascular system including atherosclerosis,calcification, thrombosis, and aneurysm; k) age-related maculardegeneration; l) AIDS; m) age- and stress-related immune systemimpairment, including impairment of tissue turnover, which occur withnatural aging, cancer, cancer therapy, acute or chronic infections,degenerative inflammatory diseases or with genetic disorders causingaccelerated cell turnover, and related anemias and other degenerativeconditions; n) healing of wounds, burns, abrasions or other acute orchronic conditions of epidermis; o) dyskeratosis congenita; p) lutealphase defect; q) premature ovarian failure (primary ovarianinsufficiency or hypergonadotropic hypogonadism); and/or r) increasingtelomerase expression and/or activity in memory T cells, therebystrengthening immune memory response and response to vaccines; s)increasing telomerase expression and/or activity in healthy tissue, thuselongating the lifespan of a subject while sustaining said subject ingood health.

Compounds of the Invention

In one embodiment, the methods of this invention comprise the use oftri-phenyl compounds represented by the structure of formula I:

wherein

Z is carbon, nitrogen, phosphor, arsenic, silicon or germanium;

R₁ to R₉ are the same or different, H, D, OH, halogen, nitro, CN,nitrileamido, amidosulfide, amino, aldehyde, substituted ketone, —COOH,ester, trifluoromethyl, amide, substituted or unsubstituted alkyl,alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl,arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl, alkylhaloalkyl,haloaryl, aryloxy, amino, monoalkylamino, dialkylamino, alkylamido,arylamino, arylamido, alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or R₃, R₄, or R₇, forms a fusedcycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with themain aromatic ring; and

R₁₀ is nothing, H, D, OH, halogen, oxo, nitro, CN, nitrileamido,amidosulfide, amino, aldehyde, substituted ketone, —COOH, ester,trifluoromethyl, amide, substituted or unsubstituted alkyl, alkenyl,alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl,alkoxy, haloalkyl, haloaryl, cycloalkyl, alkylcycloalkyl, aryloxy,monoalkylamino, dialkylamino, alkylamido, arylamino, arylamido,alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl,heterocycloalkylalkyl, heteroaryl, hetroarylalkyl, alkylheteroaryl; orits isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof, and compositionscomprising the same.

In one embodiment, the methods of this invention comprise the use oftri-phenyl compounds represented by the structure of formula II:

wherein

Z is carbon, nitrogen, phosphor, arsenic, silicon or germanium;

R₁, R₃, R₄, R₆, R₇ and R₉ are the same or different, H, D, OH, halogen,nitro, CN, nitrileamido, amidosulfide, amino, aldehyde, substitutedketone, —COOH, ester, trifluoromethyl, amide, substituted orunsubstituted alkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl,arylsulfonyl, arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl,alkylhaloalkyl, haloaryl, aryloxy, amino, monoalkylamino, dialkylamino,alkylamido, arylamino, arylamido, alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or R₃, R₄, or R₇, forms a fusedcycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with themain aromatic ring; and

R₁₀ is nothing, H, D, OH, halogen, oxo, nitro, CN, nitrileamido,amidosulfide, amino, aldehyde, substituted ketone, —COOH, ester,trifluoromethyl, amide, substituted or unsubstituted alkyl, alkenyl,alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl,alkoxy, haloalkyl, haloaryl, cycloalkyl, alkylcycloalkyl, aryloxy,monoalkylamino, dialkylamino, alkylamido, arylamino, arylamido,alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl,heterocycloalkylalkyl, heteroaryl, hetroarylalkyl, alkylheteroaryl; orits isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof, and compositionscomprising the same.

In one embodiment Z is carbon. In another embodiment R₁₀ is a methylgroup. In another embodiment R₁, R₃, R₄, R₆, R₇, and R₉ are—(CH₂)_(n)-heterocycloalkyl group, wherein n is between 1-6. In anotherembodiment R₁, R₃, R₄, R₆, R₇, and R₉ are —(CH₂)_(n)-aminoalkyl group,wherein n is between 1-6. In another embodiment R₁, R₃, R₄, R₆, R₇, andR₉ are —(CH₂)_(n)-dialkylamino group, wherein n is between 1-6 Inanother embodiment R₁, R₃, R₄, R₆, R₇, and R₉ are —(CH₂)n-N(CH₃)₂ group,wherein n is between 1-6. In another embodiment R₁, R₃, R₄, R₆, R₇, andR₉ are —(CH₂)_(n)—N(Et)₂ group, wherein n is between 1-6. In anotherembodiment R₁, R₃, R₄, R₆, R₇, and R₉ are —(CH₂)_(n)-aryl group, whereinn is between 1-6. In another embodiment R₁, R₃, R₄, R₆, R₇, and R₉ are—(CH₂)_(n)-heteroaryl group, wherein n is between 1-6. In anotherembodiment R₁, R₃, R₄, R₆, R₇, and R₉ are —(CH₂)_(n)-haloalkyl group,wherein n is between 1-6. In another embodiment R₁, R₃, R₄, R₆, R₇, andR₉ are —(CH₂)_(n)-alkoxy group, wherein n is between 1-6. In anotherembodiment R₁, R₃, R₄, R₆, R₇, and R₉ are —(CH₂)_(n)-ethoxy group,wherein n is between 1-6. In another embodiment R₁, R₃, R₄, R₆, R₇, andR₉ are —(CH₂)_(n)-cycloalkyl group, wherein n is between 1-6.

In one embodiment, the methods of this invention comprise the use oftri-phenyl compounds represented by the structure of formula III:

wherein

Z is carbon, nitrogen, phosphor, arsenic, silicon or germanium;

R′, R″ and R′″ are independently the same or different comprisinghydrogen, alkyl, haloalkyl, alkylamino, phenyl, benzyl, alkanyloyl,acetyl or benzoyl;

R₁, R₃, R₄, R₆, R₇ and R₉ are the same or different, H, D, OH, halogen,nitro, CN, nitrileamido, amidosulfide, amino, aldehyde, substitutedketone, —COOH, ester, trifluoromethyl, amide, substituted orunsubstituted alkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl,arylsulfonyl, arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl,alkylhaloalkyl, haloaryl, aryloxy, amino, monoalkylamino, dialkylamino,alkylamido, arylamino, arylamido, alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or R₃, R₄, or R₇, forms a fusedcycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with themain aromatic ring; and

R₁₀ is nothing, H, D, OH, halogen, oxo, nitro, CN, nitrileamido,amidosulfide, amino, aldehyde, substituted ketone, —COOH, ester,trifluoromethyl, amide, substituted or unsubstituted alkyl, alkenyl,alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl, arylalkylenesulfonyl,alkoxy, haloalkyl, haloaryl, cycloalkyl, alkylcycloalkyl, aryloxy,monoalkylamino, dialkylamino, alkylamido, arylamino, arylamido,alkylthio, arylthio, heterocycloalkyl, alkylheterocycloalkyl,heterocycloalkylalkyl, heteroaryl, hetroarylalkyl, alkylheteroaryl; orits isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof, and compositionscomprising the same.

In one embodiment, the methods of this invention comprise the use oftri-phenyl compounds represented by the structure of formula IV:

wherein R₁, R₃, R₄, R₆, R₇, R₉ and R₁₀ are as defined above; or itsisomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof, and compositionscomprising the same.

In one embodiment, the methods of this invention comprise the use oftri-phenyl compounds represented by the structure of formula V:

wherein

R′, R″, R′ are independently the same or different comprising hydrogen,alkyl, haloalkyl, phenyl, benzyl, alkanyloyl, acetyl or benzoyl;

R₁′, R₃′, R₄′, R₆′ R₇′, and R₉′ are the same or different comprisinghalogen, aryl, alkyl, cycloalkyl, heterocycloalkyl, alkoxy, amino, monoalkylamino, dialkylamino or arylamino group; and R₇ is as describedabove; or its isomer, pharmaceutically acceptable salt, pharmaceuticalproduct, hydrate, N-oxide, crystal or any combination thereof, andcompositions comprising the same.

In one embodiment, R₁′, R₃′, R₄′, R₆′ R₇′, and R₉′ are dialkylaminogroup. In another embodiment, R₁′, R₃′, R₄′, R₆′ R₇′, and R₉′ aredimethylamino group. In another embodiment, R₁′, R₃′, R₄′, R₆′ R₇′, andR₉′ are diethylamino group. In another embodiment, R₁′, R₃′, R₄′, R₆′R₇′, and R₉′ are N-piperidine group. In another embodiment, R₁′, R₃′,R₄′, R₆′ R₇′, and R₉′ are N-pyrolidine group. In another embodiment,R₁′, R₃′, R₄′, R₆′ R₇′, and R₉′ are N-piperazine group. In anotherembodiment, R₁′, R₃′, R₄′, R₆′ R₇′, and R₉′ are N-piperazine-4-methylgroup. In another embodiment, R₁′, R₃′, R₄′, R₆′ R₇′, and R₉′ areN-morpholine group. In another embodiment, R₁′, R₃′, R₄′, R₆′ R₇′, andR₉′ are ethoxy group.

In one embodiment, the methods of this invention comprise the use oftri-phenyl compounds represented by the structure of formula VI:

wherein R₁′, R₃′, R₄′, R₆′ R₇′, and R₉′ are the same or differentcomprising halogen, aryl, alkyl, cycloalkyl, heterocycloalkyl, alkoxy,amino, mono alkylamino, dialkylamino or arylamino group; and R₁₀ is asdescribed above; or its isomer, pharmaceutically acceptable salt,pharmaceutical product, hydrate, N-oxide, crystal or any combinationthereof, and compositions comprising the same.

In one embodiment, the methods of this invention comprise the use oftri-phenyl compounds represented by the structure of formula VII:

or its isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof, and compositionscomprising the same.

In one embodiment, the methods of this invention comprise the use oftri-phenyl compounds represented by the structure of formula VIII:

or its isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof, and compositionscomprising the same.

In one embodiment, the methods of this invention comprise the use oftri-phenyl compounds represented by the structure of formula IX:

or its isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof, and compositionscomprising the same.

In one embodiment, the methods of this invention comprise the use oftri-phenyl compounds represented by the structure of formula X:

or its isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof, and compositionscomprising the same.

In one embodiment, the methods of this invention comprise the use oftri-phenyl compounds represented by the structure of formula XI:

or its isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof, and compositionscomprising the same.

In one embodiment, the methods of this invention comprise the use oftri-phenyl compounds represented by the structure of formula XII:

or its isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof, and compositionscomprising the same.

In one embodiment, the methods of this invention comprise the use oftri-phenyl compounds represented by the structure of formula XIII:

or its isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof, and compositionscomprising the same.

In another embodiment the structure of formula I is represented by thestructure of formula XIV:

or its isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof, and compositionscomprising the same.

In another embodiment the structure of formula I is represented by thestructure of formula XV:

or its isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof, and compositionscomprising the same.

In another embodiment the structure of formula I is represented by thestructure of formula XVI:

or its isomer, pharmaceutically acceptable salt, pharmaceutical product,hydrate, N-oxide, crystal or any combination thereof, and compositionscomprising the same.

The term “alkyl” refers, in one embodiment, to a saturated aliphatichydrocarbon, including straight-chain, branched-chain and cyclic alkylgroups. In one embodiment, the alkyl group has 1-12 carbons. In anotherembodiment, the alkyl group has 1-7 carbons. In another embodiment, thealkyl group has 1-6 carbons. In another embodiment, the alkyl group has1-7 carbons. In another embodiment, the alkyl group has 2-6 carbons. Inanother embodiment, the alkyl group has 1-7 carbons. In anotherembodiment, the alkyl group has 2-8 carbons. In another embodiment, thealkyl group has 3-6 carbons. In another embodiment, the alkyl group has3-7 carbons. In another embodiment, the alkyl group has 1-4 carbons. Inanother embodiment, the branched alkyl is an alkyl substituted by alkylside chains of 1 to 5 carbons. In another embodiment, the branched alkylis an alkyl substituted by haloalkyl side chains of 1 to 5 carbons. Thealkyl group may be unsubstituted or substituted by a halogen, haloalkyl,hydroxyl, alkoxy, carbonyl, amido, alkylamido, dialkylamido, nitro,cyano, amino, monoalkylamino, dialkylamino, carboxyl, thio and/orthioalkyl.

An “alkenyl” group refers, in one embodiment, to an unsaturatedhydrocarbon, including straight chain, branched chain and cyclic groupshaving one or more double bonds. The alkenyl group may have one doublebond, two double bonds, three double bonds, etc. In another embodiment,the alkenyl group has 2-12 carbons. In another embodiment, the alkenylgroup has 2-6 carbons. In another embodiment, the alkenyl group has 2-4carbons. In another embodiment the alkenyl group is ethenyl (CH═CH₂).Examples of alkenyl groups are ethenyl, propenyl, butenyl, cyclohexenyl,etc. The alkenyl group may be unsubstituted or substituted by a halogen,hydroxy, alkoxy, carbonyl, amido, alkylamido, dialkylamido, nitro,cyano, amino, monoalkylamino, dialkylamino, carboxyl, thio and/orthioalkyl.

An alkynyl” group refers, in one embodiment, to an unsaturatedhydrocarbon, including straight chain, branched chain and cyclic groupshaving one or more triple bonds. The alkynyl group may have one triplebond, two triple bonds, triple double bonds, etc. In another embodiment,the alkynyl group has 2-12 carbons. In another embodiment, the alkynylgroup has 2-6 carbons. In another embodiment, the alkenyl group has 2-4carbons. In another embodiment the alkynyl group is ethynyl (—CH≡CH₂).Examples of alkynyl groups are ethynyl, propynyl, butynyl, cyclohexynyl,etc. The alkynyl group may be unsubstituted or substituted by a halogen,hydroxy, alkoxy, carbonyl, amido, alkylamido, dialkylamido, nitro,cyano, amino, monoalkylamino, dialkylamino, carboxyl, thio and/orthioalkyl.

An “alkoxy” group refers, in another embodiment to an alkyl group asdefined above, which is linked to oxygen. Examples of alkoxy groups areethoxy, propoxy, tert-butoxy etc.

A “haloalkyl” group refers, in one embodiment, to an alkyl group asdefined above, which is substituted by one or more halogen atoms, e.g.by F, Cl, Br or I.

An “aryl” group refers, in another embodiment, to an aromatic grouphaving at least one carbocyclic aromatic group or heterocyclic aromaticgroup, which may be unsubstituted or substituted by one or more groupsselected from halogen, haloalkyl, hydroxy, alkoxy, carbonyl, amido,alkylamido, dialkylamido, nitro, cyano, amino, monoalkylamino,dialkylamino, carboxy or thio or thioalkyl. In another embodiment, thearyl group is between 4-12-membered ring(s). In another embodiment, thearyl group is between 6-18-membered ring(s). In another embodiment, thearyl group is between 4-8-membered ring(s). In another embodiment, thearyl group is a 6-membered ring. In another embodiment, the aryl groupis a fused ring system comprising of between 2-3 rings. Nonlimitingexamples of aryl rings are phenyl, naphthyl, pyranyl, pyrrolyl,pyrazinyl, pyrimidinyl, pyrazolyl, pyridinyl, furanyl, thiophenyl,thiazolyl, imidazolyl, isoxazolyl, and the like.

A “heteroaryl” group refers, in another embodiment, to an aromatic grouphaving at least one heterocyclic aromatic group, which may beunsubstituted or substituted by one or more groups selected fromhalogen, haloalkyl, hydroxy, alkoxy, carbonyl, amido, alkylamido,dialkylamido, nitro, cyano, amino, monoalkylamino, dialkylamino, carboxyor thio or thioalkyl. In another embodiment, the heteroaryl group isbetween 4-12-membered ring(s). In another embodiment, the heteroarylgroup is between 6-18-membered ring(s). In another embodiment, theheteroaryl group is between 4-8-membered ring(s). In another embodiment,the heteroaryl group is a 6-membered ring. In another embodiment, theheteroaryl group is a fused ring system comprising of between 2-3 rings.Nonlimiting examples of heteroaryl rings are pyrrolyl, thienyl,thiazolyl, benzothienyl, naphthothienyl, purinyl, isothiazolyl, furyl,furazanyl, isobenznzofuranyl, pyranyl, chromenyl, xanthenyl,phenoxyxanthiinyl, indolyl, isoindolyl, indolizinyl, isoindolyzinyl,benzothienyl, oxazolyl, isoxazolyl, pyrazolyl, pyridyl, pyrazinyl,pyrimidinyl, pyridazinyl, and the like.

A “hydroxyl” group refers, in one embodiment, to an OH group. In someembodiments, when R₁, R₂ or R₃ of the compounds of the present inventionis OR, then R is not OH.

In one embodiment, the term “halo” refers to a halogen, such as F, Cl,Br or I.

In another embodiment, the phrase “phenol” refers to an alcohol (OH)derivative of benzene.

An “amino” group refers to, in one embodiment, to a nitrogen atomattached by single bonds to hydrogen atoms, alkyl groups, alkenyl groupsor aryl groups as described above, as described above, or a combinationthereof. Nonlimiting examples of amino groups are NH₂, N(Me)₂, N(Et)₂,N(Ph)₂ and the like.

A “cycloalkyl” group refers, in one embodiment, to a non-aromatic,monocyclic or polycyclic ring comprising carbon and hydrogen atoms. Acycloalkyl group can have one or more carbon-carbon double bonds in thering so long as the ring is not rendered aromatic by their presence.Examples of cycloalkyl groups include, but are not limited to,(C₃-C₇)cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, and cycloheptyl, and saturated cyclic and bicyclic terpenesand (C₃-C₇)cycloalkenyl groups, such as cyclopropenyl, cyclobutenyl,cyclopentenyl, cyclohexenyl, and cycloheptenyl, and unsaturated cyclicand bicyclic terpenes. Preferably, the cycloalkyl group is a monocyclicring or bicyclic to a ring structure comprising in addition to carbonatoms, sulfur, oxygen, nitrogen or any combination thereof, as part ofthe ring. In another embodiment the cycloalkyl is a 3-12-membered ring.In another embodiment the cycloalkyl is a 6-membered ring. In anotherembodiment the cycloalkyl is a 5-7-membered ring. In another embodimentthe cycloalkyl is a 4-8-membered ring. In another embodiment, thecycloalkyl group may be unsubstituted or substituted by a halogen,haloalkyl, hydroxyl, alkoxy, carbonyl, amido, alkylamido, dialkylamido,cyano, nitro, CO₂H, amino, monoalkylamino, dialkylamino, carboxyl, thioand/or thioalkyl.

A “heterocycloalkyl” group refers, in one embodiment, to a non-aromatic,monocyclic or polycyclic ring comprising carbon and in addition tocarbon, sulfur, phosphor, oxygen or nitrogen, as part of the ring. Aheterocycloalkyl group can have one or more double bonds in the ring solong as the ring is not rendered aromatic by their presence. Examples ofheterocycloalkyl groups include, but are not limited to, piperidine,piperazine, pyrane, morpholine. Preferably, the heterocycloalkyl groupis a monocyclic ring or bicyclic to a ring structure comprising inaddition to carbon atoms, sulfur, oxygen, nitrogen or any combinationthereof, as part of the ring. In another embodiment the heterocycloalkylis a 3-12-membered ring. In another embodiment the heterocycloalkyl is a6-membered ring. In another embodiment the heterocycloalkyl is a5-7-membered ring. In another embodiment the heterocycloalkyl is a4-8-membered ring. In another embodiment, the heterocycloalkyl group maybe unsubstituted or substituted by a halogen, haloalkyl, hydroxyl,alkoxy, carbonyl, amido, alkylamido, dialkylamido, cyano, nitro, CO₂H,amino, monoalkylamino, dialkylamino, carboxyl, thio and/or thioalkyl. Inanother embodiment the heterocycloalkyl is a cyclic urea, imidazolinyl,imidazolidinyl, pyrrolinyl, pyrrolidinyl, oxazolinyl, isoxazolinyl,oxazolidinyl, oxazolidonyl, isoxazolidonyl, pyrazolinyl, pyrazolidinyl,piperidyl, piperazine, morpholinyl.

The terms “alkylalkoxy”, “alkylhaloalkyl”, “alkylaryl”,“alkylcycloalkyl”, “alkylheterocycloalkyl”, “alkylheteroaryl” and“alkylamino” refer, in one embodiment, to an alkyl group, as definedabove, linked to alkoxy, haloalkyl, aryl, cycloalkyl, heterocycloalkyl,heteroaryl or amino group, respectively. The alkoxy, haloalkyl, aryl,cycloalkyl, heterocycloalkyl, heteroaryl or amino groups are as definedhereinabove. Examples include, but are not limited to, CH₂—OEt,CH₂—N-piperidine, CH₂—N-piperazine, CH₂—N(Me)₂, etc.

In another embodiment, the fused heterocycloalkyl of formula I-IV withthe main aromatic ring forms a phenylpyrrolidone group. In anotherembodiment, the fused aryl of formula I-IV, with the main aromatic ringforms a naphthalene group. In another embodiment, the fused heteroarylof formula I-IV, with the main aromatic ring forms a quinoline orisoquinoline group.

In one embodiment, this invention provides for the use of a compound asherein described and/or, its analog, derivative, isomer, metabolite,pharmaceutically acceptable salt, pharmaceutical product, hydrate,N-oxide, prodrug, polymorph, impurity or crystal or combinations thereof

In one embodiment, the term “isomer” includes, but is not limited to,optical isomers and analogs, structural isomers and analogs,conformational isomers and analogs, and the like.

In one embodiment, the term “isomer” is meant to encompass opticalisomers of the tri-phenyl compound. It is to be understood that thepresent invention encompasses any racemic, optically-active,polymorphic, or stereoisomeric form, or mixtures thereof, which formpossesses properties useful in the treatment of telomerase expressionand/or activity conditions described herein. In one embodiment, thetri-phenyl compounds are the pure (R)-isomers. In another embodiment,the tri-phenyl compounds are the pure (S)-isomers. In anotherembodiment, the tri-phenyl compounds are a mixture of the (R) and the(S) isomers. In another embodiment, the tri-phenyl compounds are aracemic mixture comprising an equal amount of the (R) and the (S)isomers. It is well known in the art how to prepare optically-activeforms (for example, by resolution of the racemic form byrecrystallization techniques, by synthesis from optically-activestarting materials, by chiral synthesis, or by chromatographicseparation using a chiral stationary phase).

The invention includes “pharmaceutically acceptable salts” of thecompounds of this invention, which may be produced, in one embodiment,to form alkali metal salts and to form addition salts of free acids orfree bases. Suitable pharmaceutically-acceptable acid addition salts ofcompounds of this invention may be prepared from an inorganic acid orfrom an organic acid. In one embodiment, examples of inorganic acids arehydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric andphosphoric acid. In one embodiment, organic acids may be selected fromaliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic,carboxylic and sulfonic classes of organic acids, examples of which areformic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic,tartaric, citric, ascorbic, glucoronic, maleic, fumaric, pyruvic,aspartic, glutamic, benzoic, anthranilic, oxalic, p-toluenesulphonic,mesylic, salicylic, p-hydroxybenzoic, phenylacetic, mandelic, embonic(pamoic), methanesulfonic, ethylsulfonic, benzenesulfonic, sulfanilic,stearic, cyclohexylaminosulfonic, algenic, galacturonic acid. In oneembodiment, suitable pharmaceutically-acceptable base addition salts ofcompounds of this invention include metallic salts made from aluminum,calcium, lithium, magnesium, potassium, sodium and zinc or organic saltsmade from N,N′-dibenzylethyleneldiamine, choline, chloroprocaine,diethanolamine, ethylenediamine, meglumine (N-methylglucamine) andprocain. All of these salts may be prepared by conventional means fromthe corresponding compounds.

Pharmaceutically acceptable salts can be prepared, from the phenoliccompounds, in other embodiments, by treatment with inorganic bases, forexample, sodium hydroxide. In another embodiment, esters of the phenoliccompounds can be made with aliphatic and aromatic carboxylic acids, forexample, acetic acid and benzoic acid esters.

The invention also includes use of N-oxides of the amino substituents ofthe compounds described herein.

This invention provides for the use of derivatives of the compounds asherein described. In one embodiment, “derivatives” includes but is notlimited to ether derivatives, acid derivatives, amide derivatives, esterderivatives and the like. In another embodiment, this invention furtherincludes use of hydrates of the compounds as described herein. In oneembodiment, “hydrate” includes but is not limited to hemihydrate,monohydrate, dihydrate, trihydrate and the like.

This invention provides, in other embodiments, use of metabolites of thecompounds as herein described. In one embodiment, “metabolite” means anysubstance produced from another substance by metabolism or a metabolicprocess.

This invention provides, in other embodiments, use of pharmaceuticalproducts of the compounds as herein described. The term “pharmaceuticalproduct” refers, in other embodiments, to a composition suitable forpharmaceutical use (pharmaceutical composition), for example, asdescribed herein.

In some embodiments, the invention provides compositions comprising thecompound of this invention or use of the compound of this invention, forincreasing telomerase activity and/or expression in a cell or tissue;and/or treating a condition by increasing telomerase activity and/orexpression in cells or tissue of a subject; and/or treating an acute orchronic condition of the epidermis; and/or elongation of a lifespan,and/or treating age- and stress-related diseases of the vascular systemincluding atherosclerosis, calcification, thrombosis, hypertension oraneurysm; and/or treating age-related macular degeneration and/orage-related diseases of the skin; and/or treating age- andstress-related immune system impairment; and/or strengthening the immuneresponse against infection-resistant organisms.

In some embodiments, the invention provides compositions comprising thecompound of this invention and/or treating female infertility-relatedconditions including luteal phase defect or premature ovarian failure(primary ovarian insufficiency or hypergonadotropic hypogonadism);and/or diminished granulosa cell telomerase activity; and/or treatingmale infertility-related conditions including impaired sperm productionor impaired sperm delivery; and/or as an adjunct to in vitrofertilization (IVF) techniques; and/or to enhance sperm quality and/oregg quality. For example, and in some embodiments, the compounds of thisinvention prolong blastocyst viability in ex vivo culture, which in turnenhances implantation efficiency. In some embodiments, the treatment ofthe population with the compounds as herein described renders them morereceptive to other IVF therapeutics, or in some embodiments, allows forthe evaluation of combination therapies, or new compounds.

In one embodiment, this invention provides methods of treatment using acompound of this invention, or composition comprising the same, asherein described. In some embodiments, the invention provides methods ofuse of a compound of this invention for the treatment of the indicateddiseases, disorders or conditions, and includes use of compositionscomprising the same.

In one embodiment, this invention provides methods of treating,suppressing, inhibiting, reducing the severity of, reducing theincidence of, reducing pathogenesis of or delaying onset of, inter alia,(a) Alzheimer's disease; (b) Parkinson's disease; (c) Huntington'sdisease; (d) stroke; e) nerve damage, motor neuron disease, multiplesclerosis (MS), peripheral and central nervous system injury includingspinal injury and cerebral vascular incidents; (f) age-related diseasesof the skin such as dermal atrophy and thinning, elastolysis and skinwrinkling, sebaceous gland hyperplasia or hypoplasia, senile lentigo,pigmentation abnormalities, graying of hair and hair loss or thinning(baldness, alopecia), or chronic skin ulcers; (g) degenerative jointdisease; (h) osteoporosis, osteoarthritis and other degenerativeconditions of the skeletal system; (i) age- and stress related diseasesof the vascular system, including atherosclerosis, calcification,thrombosis, hypertension and aneurysm; (j) age-related maculardegeneration; (k) AIDS; (l) age- and stress-related immune systemimpairment, including impairment of tissue turnover, which occurs withnatural aging, cancer, cancer therapy, acute or chronic infections,degenerative inflammatory diseases or with genetic disorders causingaccelerated cell turnover, and related anemias and other degenerativeconditions; (m) healing of wounds, burns, abrasions or other acute orchronic conditions of epidermis; (n) dyskeratosis congenital; o) lutealphase defect; p) premature ovarian failure (primary ovarianinsufficiency or hypergonadotropic hypogonadism); q) impaired spermproduction or impaired sperm delivery; r) infection withinfection-resistant organisms, via the administration of any compound asherein described and optionally other therapeutic agents, orcompositions comprising the same.

In one embodiment, the terms “treating” or “treatment” includespreventive as well as disorder remittive treatment. The terms“reducing”, “suppressing” and “inhibiting” have their commonlyunderstood meaning of lessening or decreasing, in another embodiment, ordelaying, in another embodiment, or reducing, in another embodiment theincidence, severity or pathogenesis of a disease, disorder or condition.In embodiment, the term treatment refers to delayed progression of,prolonged remission of, reduced incidence of, or amelioration ofsymptoms associated with the disease, disorder or condition. In oneembodiment, the terms “treating” “reducing”, “suppressing” or“inhibiting” refer to a reduction in morbidity, mortality, or acombination thereof, in association with the indicated disease, disorderor condition. In one embodiment, the term “progression” refers to anincreasing in scope or severity, advancing, growing or becoming worse.The term “recurrence” means, in another embodiment, the return of adisease after a remission. In one embodiment, the methods of treatmentof the invention reduce the severity of the disease, or in anotherembodiment, symptoms associated with the disease, or in anotherembodiment, reduces the number of biomarkers expressed during disease.

In one embodiment, the term “treating” and its included aspects, refersto the administration to a subject with the indicated disease, disorderor condition, or in some embodiments, to a subject predisposed to theindicated disease, disorder or condition. The term “predisposed to” isto be considered to refer, inter alia, to a genetic profile or familialrelationship which is associated with a trend or statistical increase inincidence, severity, etc. of the indicated disease. In some embodiments,the term “predisposed to” is to be considered to refer, inter alia, to alifestyle which is associated with increased risk of the indicateddisease. In some embodiments, the term “predisposed to” is to beconsidered to refer, inter alia, to the presence of biomarkers which areassociated with the indicated disease, for example, in cancer, the term“predisposed to” the cancer may comprise the presence of precancerousprecursors for the indicated cancer.

In some embodiments, the term “reducing the pathogenesis” is to beunderstood to encompass reducing tissue damage, or organ damageassociated with a particular disease, disorder or condition. In anotherembodiment, the term “reducing the pathogenesis” is to be understood toencompass reducing the incidence or severity of an associated disease,disorder or condition, with that in question. In another embodiment, theterm “reducing the pathogenesis” is to be understood to encompassreducing the number of associated diseases, disorders or conditions withthe indicated, or symptoms associated thereto.

The term “administering”, in another embodiment, refers to bringing asubject in contact with a compound of the present invention.Administration can be accomplished in vitro, i.e. in a test tube, or invivo, i.e. in cells or tissues of living organisms, for example humans.In one embodiment, the present invention encompasses administering thecompounds of the present invention to a subject.

In one embodiment, the methods of this invention make use of thedescribed compound of this invention contacting or binding a telomeraseenzyme in an amount effective to increase telomerase activity and/orexpression and thereby mediating the described effects. In someembodiments, the methods of this invention may include the preliminarystep of identifying a cell or tissue in which an increase telomeraseactivity and/or expression is desired. The cell may be in culture, i.e.in vitro or ex vivo, or within a subject or patient in vivo. In oneembodiment, an increase in telomerase expression and/or activity in acell or tissue includes, for example, enhancement of the replicativecapacity and/or lifespan of the contacted cells.

Pharmaceutical Compositions

In some embodiments, this invention provides methods of use whichcomprise administering a composition comprising the described compounds.As used herein, “pharmaceutical composition” means a “therapeuticallyeffective amount” of the active ingredient, i.e. the compounds of thisinvention, together with a pharmaceutically acceptable carrier ordiluent. A “therapeutically effective amount” as used herein refers tothat amount which provides a therapeutic effect for a given conditionand administration regimen.

In some embodiments, this invention provides compositions which maycomprise at least one compound of this invention, in any form orembodiment as described herein. In some embodiments, the term “a” is tobe understood to encompass a single or multiple of the indicatedmaterial. In some embodiments, the term “a” or “an” refers to at leastone.

In some embodiments, any of the compositions of this invention willconsist of a compound of this invention, in any form or embodiment asdescribed herein. In some embodiments, of the compositions of thisinvention will consist essentially of a compound of this invention, inany form or embodiment as described herein.

In some embodiments, the term “comprise” refers to the inclusion of theindicated active agent, such as the compounds of this invention, as wellas inclusion of other active agents, and pharmaceutically acceptablecarriers, excipients, emollients, stabilizers, etc., as are known in thepharmaceutical industry. In some embodiments, any of the compositions ofthis invention will comprise a compound of formula I-XVI in any form orembodiment as described herein. In some embodiments, any of thecompositions of this invention will consist of a compound of formulaI-XVI, in any form or embodiment as described herein. In someembodiments, of the compositions of this invention will consistessentially of a compound of this invention, in any form or embodimentas described herein. In some embodiments, the term “comprise” refers tothe inclusion of the indicated active agent, such as the compound ofthis invention, as well as inclusion of other active agents, andpharmaceutically acceptable carriers, excipients, emollients,stabilizers, etc., as are known in the pharmaceutical industry. In someembodiments, the term “consisting essentially of” refers to acomposition, whose only active ingredient is the indicated activeingredient, however, other compounds may be included which are forstabilizing, preserving, etc. the formulation, but are not involveddirectly in the therapeutic effect of the indicated active ingredient.In some embodiments, the term “consisting essentially of” refers to acomposition, whose only active ingredient with a comparable mode ofaction, or comparable molecular target is the indicated activeingredient, however, other active ingredients may be incorporated, withsuch secondary active ingredients acting on different targets, or in apalliative capacity. In some embodiments, the term “consistingessentially of” may refer to components which facilitate the release ofthe active ingredient. In some embodiments, the term “consisting” refersto a composition, which contains a compound as herein described as theonly active ingredient and a pharmaceutically acceptable carrier orexcipient.

In another embodiment, the invention provides a composition comprising acompound of this invention, as herein described, or its prodrug, analog,isomer, metabolite, derivative, pharmaceutically acceptable salt,pharmaceutical product, polymorph, crystal, impurity, N-oxide, ester,hydrate or any combination thereof and a suitable carrier or diluent.

An active component can be formulated into the composition asneutralized pharmaceutically acceptable salt forms. Pharmaceuticallyacceptable salts include the acid addition salts, which are formed withinorganic acids such as, for example, hydrochloric or phosphoric acids,or such organic acids as acetic, oxalic, tartaric, mandelic, and thelike. Salts formed from the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The pharmaceutical compositions containing the compound of thisinvention can be administered to a subject by any method known to aperson skilled in the art, such as orally, parenterally,intravascularly, paracancerally, transmucosally, transdermally,intramuscularly, intranasally, intravenously, intradermally,subcutaneously, sublingually, intraperitoneally, intraventricularly,intracranially, intravaginally, by inhalation, rectally, intratumorally,or by any means in which the recombinant virus/composition can bedelivered to tissue (e.g., needle or catheter). Alternatively, topicaladministration may be desired for application to mucosal cells, for skinor ocular application. Another method of administration is viaaspiration or aerosol formulation.

The compositions of the present invention are formulated in oneembodiment for oral delivery, wherein the active compounds may beincorporated with excipients and used in the form of ingestible tablets,buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. The tablets, troches, pills, capsules and the like mayalso contain the following: a binder, as gum tragacanth, acacia,cornstarch, or gelatin; excipients, such as dicalcium phosphate; adisintegrating agent, such as corn starch, potato starch, alginic acidand the like; a lubricant, such as magnesium stearate; and a sweeteningagent, such as sucrose, lactose or saccharin may be added or a flavoringagent, such as peppermint, oil of wintergreen, or cherry flavoring. Whenthe dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar, or both. Syrup of elixir may contain the activecompound, sucrose as a sweetening agent methyl, and propylparabens aspreservatives, a dye and flavoring, such as cherry or orange flavor. Inaddition, the active compounds may be incorporated intosustained-release, pulsed release, controlled release or postponedrelease preparations and formulations.

In another embodiment, the compositions of this invention comprise oneor more, pharmaceutically acceptable carrier materials.

In one embodiment, the carriers for use within such compositions arebiocompatible, and in another embodiment, biodegradable. In otherembodiments, the formulation may provide a relatively constant level ofrelease of one active component. In other embodiments, however, a morerapid rate of release immediately upon administration may be desired. Inother embodiments, release of active compounds may be event-triggered.The events triggering the release of the active compounds may be thesame in one embodiment, or different in another embodiment. Eventstriggering the release of the active components may be exposure tomoisture in one embodiment, lower pH in another embodiment, ortemperature threshold in another embodiment. The formulation of suchcompositions is well within the level of ordinary skill in the art usingknown techniques. Illustrative carriers useful in this regard includemicroparticles of poly (lactide-co-glycolide), polyacrylate, latex,starch, cellulose, dextran and the like. Other illustrativepostponed-release carriers include supramolecular biovectors, whichcomprise a non-liquid hydrophilic core (e.g., a cross-linkedpolysaccharide or oligosaccharide) and, optionally, an external layercomprising an amphiphilic compound, such as phospholipids. The amount ofactive compound contained in one embodiment, within a sustained releaseformulation depends upon the site of administration, the rate andexpected duration of release and the nature of the condition to betreated suppressed or inhibited.

In one embodiment it will be desirable to deliver the compositionsdisclosed herein parenterally, intravenously, intramuscularly, or evenintraperitoneally. Such approaches are well known to the skilledartisan, some of which are further described, for example, in U.S. Pat.No. 5,543,158; U.S. Pat. No. 5,641,515 and U.S. Pat. No. 5,399,363, allof which are fully incorporated by reference. In certain embodiments,solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi.

In another embodiment, it will be preferable to include isotonic agents,for example, sugars or sodium chloride. In other embodiments, prolongedabsorption of the injectable compositions will be desirable. Prolongedabsorption of the injectable compositions can be brought about by theuse of agents delaying absorption, for example, aluminum monostearateand gelatin, in the compositions.

Parenteral vehicles include in certain embodiments sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's and fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers such as those based on Ringer'sdextrose, and the like. Preservatives and other additives may also bepresent, such as, for example, antimicrobials, antioxidants, collatingagents, inert gases and the like

In some embodiments, the compounds of this invention may be administeredat various dosages to a subject, which in one embodiment, is a humansubject. In one embodiment, the compounds of this invention areadministered at a dosage of 0.1-200 mg per day. In one embodiment, thecompound of this invention is administered at a dose of 0.1-10 mg, or inanother embodiment, 0.1-25 mg, or in another embodiment, 0.1-50 mg, orin another embodiment, 0.3-15 mg, or in another embodiment, 0.3-30 mg,or in another embodiment, 0.5-25 mg, or in another embodiment, 0.5-50mg, or in another embodiment, 0.75-15 mg, or in another embodiment,0.75-60 mg, or in another embodiment, 1-5 mg, or in another embodiment,1-20 mg, or in another embodiment, 3-15 mg, or in another embodiment,1-30 mg, or in another embodiment, 30-50 mg, or in another embodiment,30-75 mg, or in another embodiment, 100-2000 mg. In some embodiments,the compounds of this invention may be administered at differentdosages, as a function of time, or disease/symptom/condition severity,or age, or other factors, as will be appreciated by one skilled in theart.

The compounds of this invention may be administered at various dosages.In one embodiment, the compounds of this invention are administered at adosage of 1 mg. In another embodiment the compounds of this inventionare administered at a dosage of 5 mg, or in another embodiment, 3 mg, orin another embodiment 10 mg, or in another embodiment 15 mg, or inanother embodiment 20 mg, or in another embodiment 25 mg, or in anotherembodiment 30 mg, or in another embodiment 35 mg, or in anotherembodiment 40 mg, or in another embodiment 45 mg, or in anotherembodiment 50 mg, or in another embodiment 55 mg, or in anotherembodiment 60 mg, or in another embodiment 65 mg, or in anotherembodiment 70 mg, or in another embodiment 75 mg, or in anotherembodiment 80 mg, or in another embodiment 85 mg, or in anotherembodiment 90 mg, or in another embodiment 95 mg or in anotherembodiment 100 mg.

While the compounds of the invention can be administered as the soleactive pharmaceutical agent, they can also be used in combination withone or more other compound, and/or in combination with other agents usedin the treatment and/or prevention of the diseases, disorders and/orconditions, as will be understood by one skilled in the art. In anotherembodiment, the compounds of the present invention can be administeredsequentially with one or more such agents to provide sustainedtherapeutic and prophylactic effects. In another embodiment, thecompounds may be administered via different routes, at different times,or a combination thereof.

In addition, the compounds of the present invention can be used, eithersingly or in combination, in combination with other modalities forpreventing or treating conditions, diseases or disorders. In someembodiments, such other treatment modalities may include withoutlimitation, surgery, radiation, hormone supplementation, dietregulation, wound debridement, etc., as will be appropriate for thecondition being treated. These can be performed sequentially (e.g.,treatment with a compound of the invention following surgery orradiation) or in combination (e.g., in addition to a diet regimen).

The additional active agents may generally be employed in therapeuticamounts as indicated in the PHYSICIANS' DESK REFERENCE (PDR) 53rdEdition (1999), or such therapeutically useful amounts as would be knownto one of ordinary skill in the art. The compounds of the invention andthe other therapeutically active agents can be administered at therecommended maximum clinical dosage or at lower doses. Dosage levels ofthe active compounds in the compositions of the invention may be variedto obtain a desired therapeutic response depending on the route ofadministration, severity of the disease and the response of the patient.The combination can be administered as separate compositions or as asingle dosage form containing both agents. When administered as acombination, the therapeutic agents can be formulated as separatecompositions that are given at the same time or different times, or thetherapeutic agents can be given as a single composition.

The pharmaceutical composition can comprise the compounds of thisinvention alone or can further include a pharmaceutically acceptablecarrier and can be in solid or liquid form such as tablets, powders,capsules, pellets, solutions, suspensions, elixirs, emulsions, gels,creams, or suppositories, including rectal and urethral suppositories.Pharmaceutically acceptable carriers include gums, starches, sugars,cellulose materials, and mixtures thereof. The pharmaceuticalpreparation containing the compounds of this invention can beadministered to a subject by, for example, subcutaneous implantation ofa pellet; in a further embodiment, the pellet provides for controlledrelease of the compounds of this invention over a period of time. Thepreparation can also be administered by intravenous, intraarterial, orintramuscular injection of a liquid preparation, oral administration ofa liquid or solid preparation, or by topical application. Administrationcan also be accomplished by use of a rectal suppository or a urethralsuppository. The pharmaceutical composition can also be a parenteralformulation; in one embodiment, the formulation comprises a liposomethat includes a complex of a compound of this invention.

The pharmaceutical composition of the invention can be prepared by knowndissolving, mixing, granulating, or tablet-forming processes. For oraladministration, the compounds of this invention or their physiologicallytolerated derivatives such as salts, esters, N-oxides, and the like aremixed with additives customary for this purpose, such as vehicles,stabilizers, or inert diluents, and converted by customary methods intoa suitable form for administration, such as tablets, coated tablets,hard or soft gelatin capsules, aqueous, alcoholic or oily solutions.Examples of suitable inert vehicles are conventional tablet bases suchas lactose, sucrose, or cornstarch in combination with binders likeacacia, cornstarch, gelatin, or with disintegrating agents such ascornstarch, potato starch, alginic acid, or with a lubricant such asstearic acid or magnesium stearate. Examples of suitable oily vehiclesor solvents are vegetable or animal oils such as sunflower oil orfish-liver oil. Preparations can be effected both as dry and as wetgranules. For parenteral administration (subcutaneous, intravenous,intraarterial, or intramuscular injection), the compounds of thisinvention or their physiologically tolerated derivatives such as salts,esters, N-oxides, and the like are converted into a solution,suspension, or emulsion, if desired with the substances customary andsuitable for this purpose, for example, solubilizers or otherauxiliaries. Examples are: sterile liquids such as water and oils, withor without the addition of a surfactant and other pharmaceuticallyacceptable adjuvants. Illustrative oils are those of petroleum, animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil, ormineral oil. In general, water, saline, aqueous dextrose and relatedsugar solutions, and glycols such as propylene glycols or polyethyleneglycol are preferred liquid carriers, particularly for injectablesolutions.

The preparation of pharmaceutical compositions which contain an activecomponent is well understood in the art. Typically, such compositionsare prepared as an aerosol of the polypeptide delivered to thenasopharynx or as injectables, either as liquid solutions orsuspensions, however, solid forms suitable for solution in, orsuspension in, liquid prior to injection can also be prepared. Thepreparation can also be emulsified. The active therapeutic ingredient isoften mixed with excipients which are pharmaceutically acceptable andcompatible with the active ingredient. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol, or the like andcombinations thereof. In addition, if desired, the composition cancontain minor amounts of auxiliary substances such as wetting oremulsifying agents, or pH buffering agents which enhance theeffectiveness of the active ingredient.

For topical administration to body surfaces using, for example, creams,gels, drops, and the like, the compounds of this invention or theirphysiologically tolerated derivatives such as salts, esters, N-oxides,and the like are prepared and applied as solutions, suspensions, oremulsions in a physiologically acceptable diluent with or without apharmaceutical carrier.

In another embodiment, the active compound can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid).

In one embodiment, the present invention provides combined preparations.In one embodiment, the term “a combined preparation” defines especiallya “kit of parts” in the sense that the combination partners as definedabove can be dosed independently or by use of different fixedcombinations with distinguished amounts of the combination partnersi.e., simultaneously, concurrently, separately or sequentially. In someembodiments, the parts of the kit of parts can then, e.g., beadministered simultaneously or chronologically staggered, that is atdifferent time points and with equal or different time intervals for anypart of the kit of parts. The ratio of the total amounts of thecombination partners, in some embodiments, can be administered in thecombined preparation. In one embodiment, the combined preparation can bevaried, e.g., in order to cope with the needs of a patient subpopulationto be treated or the needs of the single patient which different needscan be due to a particular disease, severity of a disease, age, sex, orbody weight as can be readily made by a person skilled in the art.

It is to be understood that this invention is directed to compositionsand combined therapies as described herein, for any disease, disorder orcondition, as appropriate, as will be appreciated by one skilled in theart. Certain applications of such compositions and combined therapieshave been described hereinabove, for specific diseases, disorders andconditions, representing embodiments of this invention, and methods oftreating such diseases, disorders and conditions in a subject byadministering a compound as herein described, alone or as part of thecombined therapy or using the compositions of this invention representadditional embodiments of this invention.

Treatment of Conditions or Diseases Capable of being Affected byTelomerase Activation and/or Expression

In some embodiments, this invention provides compounds, andpharmaceutical compositions comprising the same for the treatment ofconditions and/or diseases capable of being affected by enhancedtelomerase expression and/or activity. These compounds interact with thetelomerase enzyme and stimulate and/or increase telomerase expressionand/or activity in the tissues and cells of a subject. In someembodiment, such activity is decreased or absent, resulting indevelopment of, or enhanced pathogenesis or symptoms associated with adisease, disorder or condition in the subject. Such disease, disorder orcondition may comprise, inter alia, a) Alzheimer's disease; b)Parkinson's disease; c) Huntington's disease; d) stroke; e) nervedamage, motor neuron disease, multiple sclerosis (MS), peripheral andcentral nervous system injury including spinal injury and cerebralvascular incidents; f) age-related diseases of the skin such as dermalatrophy and thinning, elastolysis and skin wrinkling, sebaceous glandhyperplasia or hypoplasia, senile lentigo, pigmentation abnormalities,graying of hair and hair loss or thinning (baldness, alopecia), orchronic skin ulcers; g) degenerative joint disease; h) osteoporosis,osteoarthritis and other degenerative conditions of the skeletal system;i) age- and stress-related diseases of the vascular system includingatherosclerosis, calcification, thrombosis, hypertension and aneurysm;j) age-related macular degeneration; k) AIDS; l) age- and stress-relatedimmune system impairment, including impairment of tissue turnover, whichoccur with natural aging, cancer, cancer therapy, acute or chronicinfections, degenerative inflammatory diseases or with genetic disorderscausing accelerated cell turnover, and related anemias and otherdegenerative conditions; m) healing of wounds, burns, abrasions or otheracute or chronic conditions of epidermis; n) dyskeratosis congenita; o)sarcopenia and/or other muscular disease or condition; p) luteal phasedefect; q) premature ovarian failure (primary ovarian insufficiency orhypergonadotropic hypogonadism); r) impaired sperm production; s)impaired sperm delivery; and/or t) increasing telomerase expressionand/or activity in memory T cells, thereby strengthening immune memoryresponse and response to vaccines; and/or u) increasing telomeraseexpression and/or activity in healthy tissue, thus elongating thelifespan of a subject while sustaining said subject in good healthand/or other clinical therapeutic and/or diagnostic areas, including anyembodiment of what is encompassed by the term “treating” as describedherein.

In some embodiments, the invention provides a method of increasingtelomerase expression and/or activity in a cell or tissue, by contactingthe cell or tissue with a composition comprising a compound of thisinvention. In one embodiment, the methods may include the step ofidentifying a cell or tissue in which an increase in telomeraseexpression and/or activity is desired.

Telomerase is typically detected at low levels in normal somatic cells.In skin, lymphocytic tissues, endometrial tissue, hair follicles andintestinal crypts, active mitotic cells and stem cells, telomeraseexpression and/or activity is expressed at low levels. Telomeraseexpression and/or activity is regulated at different molecular levels,including transcription, mRNA splicing, and via the maturation andmodification of telomere reverse transcriptase (TERT) and telomere RNAcomponent (TERC).

In one embodiment, the compounds and compositions of this inventionactivate telomerase, and methods as described herein are useful thereby.

The ability of a compound to increase telomerase expression and/oractivity in a cell can be determined using the TRAP (Telomeric RepeatAmplification Protocol) assay, which is known in the art (e.g., Kim etal., U.S. Pat. No. 5,629,154; Harley et al., U.S. Pat. No. 5,891,639).The activity is typically compared to the activity similarly measured ina control assay of such cells (e g., a telomerase activity 50% greaterthan observed in a solvent control). Cell lines suitable for use in theassay, may comprise normal human fibroblasts (Now) or normal humankeratinocytes (NHK).

In some embodiments, the telomere length may serve as useful indicatorfor the telomerase expression and/or activity. In one embodiment,telomerase expression and/or activation is important in treating cancer,premature aging syndrome or segmental progeria, genetic anomalies andage-related diseases. The telomere length has distinct patterns ofexpression in specific disease progression, and is a function of itsactivation, thus measuring such length as a function of treatment hasvalue, in some embodiments, in terms of the prognosis of differentdiseases.

In one embodiment, telomere length can be measured by Southern blot,hybridization protection assay, fluorescence in situ hybridization, flowcytometry, primed in situ, quantitative-polymerase chain reaction andsingle telomere length analysis, which are all techniques known in theart (Kah-Wai Lin and Ju Yan, J. Cell. Mol. Med., 2005, Vol 9, No. 4,977-989).

In some embodiments, this invention provides methods of treating acondition capable of being affected by telomerase expression and/oractivation in a subject, the method comprising administering to asubject an effective amount of a compound of this invention, wherein thecompound stimulates or enhances telomerase expression and/or activity inthe cells or tissue of the subject.

In one embodiment, such conditions may comprise, for example, conditionsassociated with cellular senescence or with an increased rate ofproliferation of a cell in the absence of telomerase, which leads toaccelerated telomere repeat loss. The term “increased rate ofproliferation” refers to higher rate of cell division compared to normalcells of that cell type, or compared to normal cells within otherindividuals of that cell type. The senescence of those groups of cellsat an abnormally early age can eventually lead to disease.

In some embodiments, conditions which may be treated by increasingtelomerase expression and/or activity may comprise use of the compoundsas described herein in ex vivo cell therapy, as described further below,employing the appropriate associated cell types. In one embodiment, thecondition is Alzheimer's disease, Parkinson's disease, Huntington'sdisease, stroke, nerve damage, motor neuron disease, multiple sclerosis(MS), or peripheral and central nervous system injury including spinalinjury and cerebral vascular incidents; wherein the cells that may beemployed are cells of the central nervous system, including neuronsand/or glial cells, e.g., astrocytes, endothelial cells and/orfibroblasts. In another embodiment, the condition is glutamate-inducedapoptosis in the cerebellum.

In another embodiment, the condition is an age-related disease of theskin, such as dermal atrophy and thinning, elastolysis and skinwrinkling, sebaceous gland hyperplasia or hypoplasia, senile lentigoand/or other pigmentation abnormalities, graying of hair and hair lossor thinning (baldness, alopecia), or chronic skin ulcers; wherein thecells that may be employed are fibroblasts, sebaceous gland cells,melanocytes, keratinocytes, Langerhan's cells, microvascular endothelialcells and/or hair follicle cells.

In another embodiment, the condition is a degenerative joint disease;wherein the cells that may be employed are cells of the articularcartilage, such as chondrocytes and lacunal and/or synovial fibroblasts.

In another embodiment, the condition is osteoporosis, osteoarthritisand/or other degenerative conditions of the skeletal system; wherein thecells that may be employed are cells of the skeletal system, such asosteoblasts, bone marrow stromal or mesenchymal cells and/orosteoprogenitor cells.

In another embodiment, the condition is sarcopenia and/or otherdegenerative conditions of the musculature; wherein the cells that maybe employed are cells are muscle cells, or progenitor cells thereof ormesenchymal cells.

In another embodiment, the condition is an age- and/or stress-relateddisease of the vascular system including atherosclerosis, calcification,thrombosis, hypertension and aneurysms; wherein the cells that may beemployed are cells of the heart and vascular system, includingendothelial cells, smooth muscle cells, and/or adventitial fibroblasts.

In another embodiment, the condition is age-related maculardegeneration; wherein the cells that may be employed are cells of theeye, such as pigmented epithelium and/or vascular endothelial cells.

In another embodiment, the condition is acquired immune deficiency orAIDS; wherein the cells that may be employed are T lymphocytes, such asCD4+ or CD8+ T cells. In another embodiment, the condition is congenitalimmune deficiency.

In another embodiment, the condition is graft-versus-host disease(GVHD). In another embodiment, the condition is graft-versus leukemiadisease (GVL).

In another embodiment, the condition is age- and/or stress-relatedimmune system impairment, including impairment of tissue turnover, whichoccurs with natural aging, cancer, cancer therapy, acute or chronicinfections, degenerative inflammatory diseases or with genetic disorderscausing accelerated cell turnover, and related anemias and otherdegenerative conditions, wherein the cells that may be employed areother cells of the immune system, including cells in the lymphoid,myeloid, and erythroid lineages, such as B and T lymphocytes, monocytes,circulating and specialized tissue macrophages, neutrophils,eosinophils, basophils, NK cells, and their respective progenitors.

In some embodiments, the methods and/or compositions of this inventionfind application in improving immune function in disease or in health.In some embodiments, the methods and/or compositions of this inventionfind application in enhancing T cell activity, for example cytotoxic Tlymphocyte activity or responsiveness, or rescue from anergy, which mayfind application in treating multiple diseases, for example in infectionor neoplasia. In some embodiments, such methods and/or compositions areparticularly useful in treating pathogens highly resistant toconventional therapy, particularly virulent organisms, or organisms forwhich no other therapies are available, for example,multi-drug-resistant organisms.

In another embodiment, the condition is female fertility-related,including luteal phase defect, wherein there is a disruption in thenormal female menstrual cycle and the body does not produce enoughprogesterone, resulting in a delay in the development of the lining ofuterus (endometrium), or premature ovarian failure (primary ovarianinsufficiency or hypergonadotropic hypogonadism), wherein there is aloss of normal functioning of the ovaries in a woman younger than age40.

In another embodiment, the condition is male fertility-related,including impaired sperm production or impaired sperm delivery.

In another embodiment, cell types in which an increase in telomeraseexpression and/or activity can be therapeutically beneficial include,but are not limited to, cells of the liver, endocrine and exocrineglands, smooth musculature, or skeletal musculature.

In one embodiment, AIDS disease is believed to be caused by the earlysenescence of CD8+ cells. The aging of such cells is attributed notsimply to an abnormal amount of loss of telomere sequences per celldoubling, but, in addition, to the increased replicative rate of thecells, such that telomere attrition is greater than normal for thatgroup of cells. The invention thus provides methods of treating an HIVinfected subject, and more particularly of reducing early senescence ofHIV-restricted CD8+ cells in an HIV infected subject, comprisingadministering to said subject a compound of this invention or acomposition comprising the same.

In one embodiment an increase in telomerase expression and/or activitycan benefit non-dividing cells as well as proliferating cells, e.g. inconditions associated with increased susceptibility to cell death due tostress, such as ischemia in heart failure or in stroke (Schneider, J.Mol. Cell. Cardiol 34(7):717-24; Mattson, Exp Gerontol. 35(4):489-502).This invention provides methods of reducing stress- or DNAdamage-induced cell death in a subject, such as a subject experiencingischemic conditions in tissue due to heart failure or stroke, byincreasing telomerase expression and/or activity in cells of thesubject, comprising administering to said subject a compositioncomprising a compound of this invention.

In one embodiment, this invention provides methods for extending thelifespan of a subject and whose life can be extended by extending theability of those cells to continue replication or resist stress-inducedcell death. One example of such a group of cells is lymphocytes presentin Down's Syndrome patients. The invention thus provides a method ofenhancing replicative capacity and/or lifespan of lymphocytes present ina Down's Syndrome patient, by increasing telomerase expression and/oractivity in said cells of the patient, comprising administering to saidsubject a composition comprising a compound of this invention. Thecompositions may also be used to improve resistance to stress-inducedcell death occurring during normal aging.

In one embodiment, this invention provides methods of increasingtelomerase expression and/or activity and thereby promote healing ofwounds, burns, abrasions or other acute or chronic conditions of theskin, and in some embodiment, in particular in the epidermis. Theinvention thus provides a method of treating an acute or chroniccondition of the skin, by administering to said subject a compound asherein described and/or a composition comprising the same. In oneembodiment, the composition is topically administered to the affectedarea.

In another embodiment, acute or chronic skin conditions treated via themethods of this invention may comprise lesions suffered in trauma,burns, abrasions, surgical incisions, donor graft sites, and/or lesionscaused by infectious agents.

In another embodiment, the acute or chronic conditions of the skin maycomprise chronic venous ulcer, diabetic ulcer, compression ulcer,pressure sores, and ulcers or sores of a mucosal surface.

In another embodiment, the acute or chronic condition of the skin maycomprise surface lesions caused by a persistent inflammatory conditionor infection, or by a genetic defect (such as Haloid formation andcoagulation abnormalities).

In one embodiment, the methods of healing of wounds, burns, abrasions orother acute or chronic conditions of the skin comprises administering acomposition comprising a compound as herein described to stimulate orenhance cell proliferation or migration at the treatment site, increasein density of epithelial cells at the site as a result of the appliedtherapy and thereby closure of a wound if present, or restoration ofnormal physiological function.

In one embodiment, this invention contemplates manipulation of the skinand repair of any imperfection of the skin surface for other purposes,such as cosmetic enhancement.

In another embodiment, compounds and compositions as herein describedmay be utilized for protecting skin from UV radiation, or palliativetreatment of damage thereby.

In another embodiment, the methods and compositions of the invention canbe used in treating hair conditions, in preserving hair color, hairshine or quality. In some embodiments, methods and compositions of theinvention can be used in treating hair loss, or as adjunctive therapywith hair replacement and hair loss treatment protocols.

In another embodiment, the methods and compositions of the invention canbe used in agricultural applications. In one embodiment, compounds andcompositions as herein described may be utilized to increase plant cropyields, wherein, in one embodiment, fruit yield is increased and inanother embodiment, fruit size is increased. In another embodiment, thecompounds and compositions as herein described may be utilized toincrease milk production in dairy cattle, while in another embodiment,egg size may be increased. In another embodiment, meat production may beincreased in domestic animals raised for meat, including chickens, cowsand pigs. In yet another embodiment, the compounds and compositions asherein described may be utilized in the horse racing industry, toincrease reproductive ability of both stallions and mares, and toincrease muscle strength and stamina for greater racing speeds.

In one embodiment, the methods and compositions of the invention can beused to enhance replicative capacity and/or extend lifespan of cells inculture, e.g., in ex vivo cell therapy or in monoclonal antibodyproduction, by increasing telomerase expression and/or activity in thecells. Increasing telomerase expression and/or activity increases thereplicative capacity of such cells by slowing telomere repeat lossand/or improving resistance to stress-induced cell death during cellproliferation.

In one embodiment, the methods and compositions of the invention may beuseful in allogeneic cell therapy, which may include, in one embodiment,stem cell transplantation, donor cell transplantation or cancerimmunotherapy. In another embodiment, the methods and compositions ofthe invention may be useful in the activation and/or mobilization ofstem cells. For example, and in some embodiments, the methods andcompositions of the invention may be useful in enhancing survival ofstem cells in ex vivo culture. In some embodiments, the methods andcompositions of the invention may prolong culture time, therebysupporting expansion of the cell population, which in turn enhancestransplantation efficiency, while in other embodiments, the methods andcompositions of the invention may increase cell viability and/or mayincrease lifespan of the cells at the stem cell stage, prior to celldifferentiation. In some embodiments, the treatment of the populationwith the compounds as herein described renders them more receptive toother stem cell manipulations, for example transformation ortransduction.

In one embodiment, the stem cells may be embryonic stem cells, while inanother embodiment; the stem cells may be adult stem cells. In oneembodiment, the methods and compositions of the invention may be usefulfor culturing stem cells in vitro. In another embodiment, the methodsand compositions of the invention may be useful in culturing non-stemcells in vitro. In one embodiment, the methods and compositions of theinvention may be useful for culturing, inter alia, pancreatic beta cellsin vitro. In another embodiment, the methods and compositions of theinvention may be useful for tissue engineering. In another embodiment,the stem cells may be cancer stem cells. In one embodiment, the methodsand compositions of the invention may be useful in treating aplasticanemia.

In some embodiments, the methods and compositions of this invention areuseful as an adjunctive therapy in treating cancer, in combination withsurgical, radiation, chemotherapeutic and immunotherapeuticmethods/compounds, and in enhancing responsiveness to other cancertherapeutics. In some embodiments, the compounds of this inventionsuppress cancer latency, i.e. maintain neoplastic cells in an activestate whereby additional compounds which are toxic to cancer cells aremore effective. In some embodiments, the methods and compositions ofthis invention when comprising adjunctive therapy for cancer increasethe likelihood of survival of a subject with cancer. In someembodiments, the methods and compositions of this invention may preventdamage to developing embryos caused by drug treatment, which may, in oneembodiment, may be anti-cancer drug treatment. In some embodiments, themethods and compositions of this invention are useful in the developmentof cancer diagnostics. For example, and in some embodiments, thecompounds of this invention expand populations of hard to detect cancercells, aiding in their detection, thereby being a means/method of cancerdiagnostics. In some embodiments, staging of the cancer may be areflection of the responsiveness to the compounds as herein described.

In some embodiments, the methods and compositions of this invention areuseful in the development of novel treatment regimens. For example, andin some embodiments, the compounds of this invention expand populationsof difficult to detect or difficult to treat cancer cell populations,which in turn allows for the design of anticancer compounds moresuitable for the treatment of the particular cancerous cell population.In some embodiments, the treatment of the population with the compoundsas herein described renders them more susceptible to other anticancertherapeutics, or in some embodiments, allows for the evaluation ofcombination therapies, or new compounds.

In some embodiments, the methods and compositions of this invention areuseful in cancer stem cell expansion, which in turn is useful in thedevelopment of therapies and treatments for cancers comprising suchcells. In some embodiments, the methods and compositions of thisinvention useful in cancer stem cell expansion, disease staging anddiagnosis of the cancer.

In one embodiment, the methods and compositions of the invention may beuseful in facilitating transdifferentiation of cells, while in anotherembodiment, they may be useful in cell cloning and/or in transduction ortransformation. In another embodiment, the methods and compositions ofthe invention may be useful in enhancing plasmid or naked DNA uptake,liposome uptake, etc., in cells, resulting in some embodiments, inenhanced nucleic acid transformation efficiency.

In one embodiment, this invention provides methods for increasing thereplicative capacity and/or lifespan of the cells by ex vivoapplications, wherein a compound of this invention is added to explantcells obtained from a subject, thereby increasing the replicativecapacity and/or lifespan of the cells.

The explant cells may include, for example, stem cells, such as bonemarrow stem cells (U.S. Pat. No. 6,007,989), bone marrow stromal cells(Simonsen et al., Nat Biotechnol 20(6):592-6, 2002), or adrenocorticalcells (Thomas et al., Nat Biotechnol 18(1):39-42, 2000). Diseaseconditions of this invention may also be subject to ex vivo cell-basedtherapy. Examples include the use of muscle satellite cells fortreatment of muscular dystrophy, osteoblasts to treat osteoporosis,retinal pigmented epithelial cells for age-related macular-degeneration,chondrocytes for osteoarthritis, etc.

In one embodiment, this invention provides methods for treatingdyskeratosis congenita, comprising administering a compound acomposition comprising a compound of this invention.

Dyskeratosis congenita (DKC), also known as Zinsser-Engman-Colesyndrome, is a rare, progressive bone marrow failure syndromecharacterized by the triad of reticulated skin hyperpigmentation, naildystrophy, and oral leukoplakia. The gene mutated in X-linked DKC (DKC1)encodes a highly conserved nucleolar protein called dyskerin. Evidenceexists for telomerase dysfunction, ribosome deficiency, and proteinsynthesis dysfunction in this disorder. Early mortality is oftenassociated with bone marrow failure, infections, fatal pulmonarycomplications, or malignancy.

Patients with DKC have reduced telomerase expression and/or activationand abnormally short tracts of telomeric DNA compared with normalcontrols. Because telomeres function to maintain chromosomal stability,telomerase has a critical role in preventing cellular senescence andcancer progression. Both DKC and a subset of aplastic anemia are due toa defect in telomerase.

In one embodiment, the invention provides a method of stem cellproliferation, wherein a stem cell population is treated with a compoundof this invention and thereby enhances the replicative capacity and/orlifespan of the cell population.

In some embodiments, the invention provides for the use of compoundsand/or compositions as herein described as therapeutic agents toforestall and reverse cellular senescence, including but not limited toconditions associated with cellular senescence. In some embodiments, thecompounds and/or compositions as herein described may be used in cell ororgan culture media, to expand primary cell cultures for purposes of,inter alia, diagnosis, analysis or vaccine production, or to maintainthe viability of an organ/cells prior to transplantation (duringischemic time, the time between the interruption and reestablishment ofblood supply). In some embodiments, the invention provides for the useof compounds and/or compositions as herein described are useful inprolonging survival of cells or tissue for transplantation, and/or insome embodiments, promote greater incorporation of such grafts. In someembodiments, such properties find utility in particular in applicationssuch as islet or marrow cell transplantation, stem cell transplantationor tissue engineering.

In some embodiments, the invention provides for the use of compoundsand/or compositions as herein described as stimulants of (a) cells withreplicative capacity in the central nervous system, includingastrocytes, endothelial cells, and fibroblasts which play a role in suchage-related diseases as Alzheimer's disease, Parkinson's disease,Huntington's disease, and stroke, or in repairing nerve damage, motorneuron disease, peripheral and central nervous system injury includingspinal injury and cerebral vascular incidents (CVI) such as stroke, orin diseases such as multiple sclerosis (MS); (b) cells with finitereplicative capacity in the integument, including fibroblasts, sebaceousgland cells, melanocytes, keratinocytes, Langerhans cells, and hairfollicle cells which may play a role in age-related diseases of theintegument such as dermal atrophy, elastolysis and skin wrinkling,sebaceous gland hyperplasia, senile lentigo, graying of hair and hairloss (baldness, alopecia), chronic skin ulcers, keratosis andage-related impairment of wound healing; (c) cells with finitereplicative capacity in the articular cartilage, such as chondrocytesand lacunal and synovial fibroblasts which play a role in degenerativejoint disease; (d) cells with finite replicative capacity in the bone,such as osteoblasts and osteoprogenitor cells which play a role inosteoporosis; (e) cells with finite replicative capacity in the immunesystem such as B and T lymphocytes, monocytes, neutrophils, eosinophils,basophils, NK cells and their respective progenitors, which may play arole in age-related immune system impairment; (f) cells with finitereplicative capacity in the vascular system including endothelial cells,smooth muscle cells, and adventitial fibroblasts which may play a rolein age-related diseases of the vascular system includingatherosclerosis, calcification, thrombosis, hypertension and aneurysms;(g) cells with finite replicative capacity in body organs including, butnot limited to liver, lung and pancreas (islet cells), which may play arole in liver disease (cirrhosis), lung disease or diabetes; (h) cellswith finite replicative capacity in the reproductive system, includingovarian follicle cells and corpus luteum cells; (i) cells with finitereplicative capacity in the ear, including inner and outer ciliatedauditory cells of the organ of Corti; and (j) cells with a finitereplicative capacity in the eye such as pigmented epithelium andvascular endothelial cells which may play an important role inage-related macular degeneration.

The following examples are presented in order to more fully illustratethe preferred embodiments of the invention. They should in no way beconstrued, however, as limiting the broad scope of the invention.

EXAMPLES Example 1 Synthesis of Compound 77

1,1,1-tris(4-hydroxyphenyl)ethane (4 g, 13 mM), formaldehyde (3.6 g, 120mM) and a 40% solution of dimethylamine in water (15 ml) were added to asolution of 50 ml water and 60 ml EtOH. The solution was refluxed for2.5 hours. Partial evaporation of the solvent precipitated a whitesolid, which was filtered, washed with water and dried to give 7.85 gwhite solid of compound 77, 93% yield, mp.=169°.

NMR CDCl₃ δ 6.64 (6H, s, ArH), 3.40 (12H, s, CH₂), 2.22 (36H, s, N—CH₃),2.06 (3H, s, C—CH₃).

Example 2 Synthesis of Compound 84

Compound 84 was synthesized by a process comparable to that described inExample 1.

NMR CDCl₃ δ 6.71 (6H, s, ArH), 3.58 (12H, s, CH₂), 2.54 (24H, q, J=7.0Hz), 1.04 (24H, t, J=7.0 Hz).

Example 3 Synthesis of Compound 78

1,1,1-tris(4-hydroxyphenyl)ethane (1.53 gr, 5 mM), formaldehyde (1.35gr, 45 mM) and 1-methyl piperazine (2.5 ml, 50 mM) in 20 ml water and 25ml EtOH were refluxed for 3 hours. Evaporation provided a solid that byTLC and NMR contained 2 products, which was not the starting material.Formaldehyde (0.75 gr, 25 mM) and 1-methyl piperazine (1.5 ml, 30 mM)were added to 5 ml water and 10 ml EtOH and the reaction was refluxedfor 4 hours. Evaporation and workup gave 3.3 gr light yellow-whitesolid, 67% yield, mp. 63°. Soluble in ethanol, and very good solubilityin water.

NMR CDCl₃ δ 6.67 (6H, s, ArH), 3.53 (12H, s, CH₂), 2.44 (48H, br.m, ringpiperazine), 2.26 (18H, s, N—CH₃), 2.00 (3H, s, C—CH₃).

Example 4 Synthesis of Compound 83

Compound 83 was synthesized by a process comparable to that described inExample 1. A white solid was obtained. mp.=178°.

NMR CDCl₃ δ 6.68 (6H, s, ArH), 3.55 (12H, s, CH₂), 2.51 (24H, br.t,N—CH₂ ring), 2.03 (3H, s, C—CH₃), 1.55 (24H, br.t, N—CH₂ ring), 1.42(12H, br.s).

Example 5 Synthesis of Compound 81

Compound 81 was synthesized by a process comparable to that described inExample 1. A white solid was obtained. m.p.=135°.

NMR CDCl₃ δ 6.68 (6H, s, ArH), 3.61 (12H, s, CH₂), 2.51 (24H, br.t,N—CH₂ ring), 2.03 (3H, s, C—CH₃), 1.76 (24H, br.t, N—CH₂ ring).

Example 6 Synthesis of Compound 82

Compound 82 was synthesized by a process comparable to that described inExample 1. A white solid was obtained. mp.=212°.

NMR CDCl₃ δ 6.68 (6H, s, ArH), 3.69 (24H, t, J=4.5 Hz, N—CH₂ ring), 3.52(12H, s, CH₂), 2.45 (24H, br.t, O—CH₂ ring), 2.03 (3H, s, C—CH₃).

Example 7 Synthesis of Compound 79 Step 1

Compound 77 (2.98 g, 4.6 mM), prepared by a process as described inExample 1, was added to 20 ml acetic anhydride, and heated to 100° for 4hours. The mixture was cooled and water was added. The mixture wasstirred overnight at room temperature, and then extracted with CH₂Cl₂.The solvent was evaporated to give a nona-acetate derivative as yellowoil and was further purified by chromatography (silica gel; 1%MeOH/CH₂Cl₂) to give 3.2 g of viscous yellow oil, 80% yield.

Step 2

A KOH (4 g) solution in water was added to a solution of thenona-acetate of step 1 (2.5 g) in 20 ml EtOH. The mixture was stirredfor 20 hours at room temperature. The mixture was acidified with HCl,and extracted with CH₂Cl₂. The solvent was evaporated and gave 2.2 g ofa yellow oil that and was further purified by column chromatography(silica gel; 2% MeOH/CH₂Cl₂) and recrystallyzed from toluene-hexane togive 1 gr of compound 79, 53% yield, white solid, mp 78°. TLC-Rf=0.55 in5% MeOH/CH₂Cl₂.

NMR CDCl₃ δ 7.93 (3H, s, OH), 6.79 (6H, s, Ar—H), 4.54 (12H, s, Ar—CH₂),3.55 (12H, q, J=7.0 Hz, CH₂), 2.05 (3H, s, C—CH₃), 1.22 (18H, t, J=7.0Hz, CH₃).

Example 8 Synthesis of 1,1,1-tris(4-hydroxy-3,5-dibromo-phenyl)-ethane(Compound 68) Step 1

A solution of NaOH (1 g, 25 mM) in 10 ml water and dimethyl sulphate(5.1 gr, 40 mM)(1:8 molar ratio) was added during 1 hour andsimultaneously in portions to a solution of1,1,1-tris(4-hydroxyphenyl)-ethane (1.53 g, 5 mM) in 20 ml ethanol and10 ml water. The solution was then refluxed for 1 hour, and stirred 70hours at RT. The white precipitate was filtered, washed with water anddried to give 1.74 g of 1,1,1-tris(4-methoxyphenyl)-ethane.Recrystalization twice from 50 ml ethanol gave 1.15 gr white crystals,66% yield, and m.p. 160°. TLC Rf=0.85 in CH₂Cl₂.

NMR CDCl₃ δ 6.99, 6.79 (12H, AB_(q), J_(AB)=8.8 Hz), 3.78 (9H, s, OCH₃),2.11 (3H, s, CH₃).

Step 2

To a solution of 1,1,1-tris(4-methoxyphenyl)-ethane (0.49 gr, 1.4 mM,),from step 1, in 22 ml 1,2-dichloroethane, a solution of bromine (1.65gr, 10.2) (7.3:1 ratio) in 5 ml 1,2-dichloroethane was added inportions. The solution was stirred at RT overnight and heated for 3hours to 70°, and worked up (sodium thiosulphate) to give 1.0 gr crudeproduct. TLC shows no starting material, but NMR showed mixtures,indicating that the bromination was not complete (m at 6.90 ppm, and 4methoxy). The solid was brominated again with 1 gr bromine and refluxed18 hours. The mixture was worked up as above and triturated with hotethanol to give 0.27 gr white solid, 23% yield, mp=160°. TLC Rf=0.95 inCH₂Cl₂.

NMR CDCl₃ δ 7.16 (6H, s, ArH), 3.92, 3.91 (6:4 ratio)(9H, 2s, OCH₃),2.04, 2.03 (4:6 ratio)(3H, s, CH₃).

Example 9 Synthesis of 1,1,1-tris(4-hydroxy-3,5-diiodo-phenyl)-ethane

To 1,1,1-tris(4-hydroxyphenyl)-ethane (1.53 g, 5 mM) in 40 ml ethanoland 40 ml water cooled in ice, KOH (2.2 gr, 39.2 mM) followed by KI (5.8g, 34.8 mM) and iodine (8.8 g, 34.7 mM) were added. The color turns fromviolet to brown. The reaction was stirred at room temperature for 3hours. The mixture was added to crushed ice. Concentrated HCl was addedto obtain acidic pH and was treated with thiosulphate solution andworked up. Evaporation gave 5.1 g light brown solid, hexa iodo productfollowed by trituration in ethanol gave 3 g white solid, 61% yield,mp=230°. RF=0.8 (in 5% MeOH—CH₂Cl₂).

NMR CDCl₃ δ 7.3 (6H,$), 5.77 (br.s, OH), 1.97 (3H, s, CH₃).

Example 10 Repair of DNA Double-Stranded Fragments Using Telomerase

In order to assess telomerase activation and/or expression, a DNA repairassay was conducted. DNA fragments were generated by ionic radiation.All the compounds increased repair. These results indicate thattelomerase activity and/or expression provided stability in stressconditions.

Example 11 Effects of Tri-Phenyl Telomerase Activating Compounds onLifespan Extension with Nematodes

Nematodes (C. elegans) were administered tri-phenyltelomerase-activating compounds presented in the following tables, andtelomerase activity and/or expression was measured in terms of nematodelifespan extension. Nematodes were grown with the activators at 50micromolar concentration, and the mean, maximum and half-life forCompound 68 and Compound 77, two representative molecules, was measuredversus that of the control (FIG. 8). Values represent mean lifespan.

TABLE 1

Compound Lifespan # R₁ R₂ R₃ R₄ ext. %  1 OH OH OH CH₃   30  2 OAc OAcOAc CH₃  3 H H H Cl  4 H H H CH₃  5 OCH₃ OCH₃ OCH₃ CH₃   12  6 OCH₃ OCH₃H CH₃  7 OCH₃ H H OH  8 Cl  9 CH₃ 10 CH₃ H H CH₃  −1 11 OCH₃ CH₃ CH₃ OH12 Cl 13 CH₃ 14 OCH₃ OCH₃ CH₃ OH 15 Cl 16 CH₃ 17 OCH₃ OCH₃ F OH 18 Cl 19CH₃ 20 CH₃ CH₃ CH₃ OH 21 Cl 22 CH₃   21 23 1-naphtyl OCH₃ OEt OH 24 Cl25 CH₃ 26 1-naphtyl 1-naphtyl 1-naphtyl OH 27 Cl 28 CH₃ 29 9-fluoreneOCH₃ OH 30 9-fluorene OCH₃ Cl 31 CH₃   23 31 9-xanthene H Cl 33 CH₃ −2634 Satu. 7 OCH₃ OH ring 35 Cl 36 CH₃ 37 Unsatu. 7 OCH₃ OH ring 38 Cl 39CH₃ 40 41 Tol Bz Tol Bz Tol Bz CH₃ 42 OCH₂COOEt OCH₂COOEt OCH₂COOEt CH₃ −7 43 OCH₂COOH OCH₂COOH OCH₂COOH CH₃ 44 H H H Cl 45 H H H CH₃ 46 H H HOEt 47 H H H O-isopropyl 48 H H H O-nbutyl 49 H H H CH₃   16 50 3-INDOL3-INDOL OCH₃ H    4 51 3-INDOL 3-INDOL OCH₃ CH₃ 52 Destetradimethylamino −25 53 4-Py OH OH CH₃   13 54 3-PY OH OH CH₃   15 554-Py H H CH₃ 56 4-Py CH₃ CH₃ CH₃ 57 4-Py OCH₃ OCH₃ CH₃ 58 4-Py OCH₃ OHCH₃ 59

Compound # R X Lifespan % 60 OH P  −2 61 OH P═O   30 62 OCH₃ P   29 63OCH₃ P═O    0 64 H P   19 65 CH₃ P   42 66 Br N   19

Compound # R₁ R₂ R₃ Lifespan % 67 Cl OH Cl 68 Br OH Br   13 69 Br OH H −5 70 Br OCH₃ Br   41 71 NO₂ OH H    8 72 NO₂ OCH₃ H    4 73 I OCH₃ I −7 74 I OH H 75 OH OH OH 76 NH₂ OH H 77 CH₂- OH CH₂-   43 DimethylaminoDimethylamino 78 CH₂- OH CH₂-   36 Methylpiperazine Methylpiperazine 79CH₂OEt OH CH₂OEt   16 80 CH₂OAc OAc CH₂OAc 81 CH₂- OH CH₂- pyrrolidinepyrrolidine 82 CH₂- OH CH₂- morpholine morpholine 83 CH₂- CH₂-piperidine piperidine 84 CH₂- CH₂- diethylamine diethylamine

Example 12 Effects of Tri-Phenyl Telomerase Activating Compounds onHuman Glioblastoma Cells

Compounds 1, 62, 68, 77 and 79 (referred to in the tables above) wereexamined for telomerase activation and/or expression in glioblastomacells. Compound 79 and 68 were synthesized as described hereinabove.Compounds 1 and 62 are commercially available. One μM of nuclear cellproteins (as the source of telomerase) were added to a specific reactionmixture of telomerase and a telomerase repeat amplification (TRAP) assaywas performed in the absence or presence of telomerase activators(Compounds 79, 68, 1 and 62). The reaction products were analyzed onPAGE followed by autoradiography. The % of telomeraseactivity/expression was calculated.

U-251 gliobastoma cells were treated with different concentrations ofcompounds of the present invention for 1 or 3 hours. The culture mediumwas removed and the cells were washed several times with PBS. Chapsnuclear extract was performed by standard methodology and telomeraseactivity was determined by TRAP using radioactive nucleotides. Arepresentative picture of several experiments demonstrating the productsof telomerase activity observed by the TRAP assay is shown in FIG. 1A.Cells were treated with Compound 68 at 1 (lanes 2 and 4), or 0.25 μM(lanes 3 and 5) for 1 (lanes 2-3) or 3 (lanes 4-5) hours, respectively.Lane 1 shows vehicle treatment only. Quantification of telomeraseactivity from multiple experiments was performed using a β-scintillationcounter by measuring the total radioactive labeling of telomeraseproducts, and telomerase activity in each sample was calculated relativeto the internal standard. The percent telomerase activation (from thecontrol cells treated with the vehicle only) was determined for thecompounds of the present invention (FIG. 1B). Compounds of the presentinvention significantly increased the activity of telomerase in treatedcells and the level of activation was dependent on the nature of thecompound, the concentration and the time of exposure of the cells to thecompounds of the present invention.

The effect of compounds of the present invention on the level oftelomerase protein and of long-term treatment of a single dose ofCompound 68 on telomerase was tested by exposing cells for variousintervals (3-24 h) to 1 μM of the compound and detecting telomeraseprotein level by western blot analysis with anti-telomerase antibody.Compound 68 increased telomerase protein level in treated cells (FIG.2A). A time-dependent increase in telomerase was observed, withactivation peaking at 1-3 h of treatment, and then gradually decreasing.The level of telomerase activity was only slightly higher compared tothat found in vehicle-treated cells after 24 h (FIG. 2B). No changes inthe level of β-actin protein (lower panel) or other nuclear enzymes suchas topoisomerase I were observed, suggesting that a single dose causes afast, specific activation of telomerase, although this activation istransient and the level of telomerase returned to its basal value 24 hafter treatment, indicating an ability of compounds of the presentinvention to control telomerase activation.

Experiments were also performed to determine the effect of compounds ofthe present invention on the telomerase mRNA level (including the fulland spliced variant telo-mRNAs). Cells were treated with a single doseof 1 mM Compound 68 for various intervals (1-24 h). Total RNA extractswere prepared, and equal RNA concentrations were analyzed by northernblotting with a specific hTERT probe. FIG. 3A shows the telomerasefull-length and splice variant mRNAs in the control untreated cells(lanes 1,2). The level of the full-length mRNA as well as the splicevariant RNAs significantly increased with time in cells treated withcompounds of the present invention (up to 4.5-fold of control) (FIG. 3B,lanes 3-7). The increased expression of telomerase was specific since noeffect on GAPDH mRNA was observed (lower panel). Addition of actinomycinD (an RNA transcription inhibitor) during treatment with compounds ofthe present invention (for 3 h) abolished the activation of telomeraseexpression (FIGS. 3A and B, compare lane 8 to 4), suggesting thatcompounds of the present invention activate the expression of thetelomerase gene.

Example 13 Effects of Compounds of the Present Invention on HumanMesenchymal Stem Cells

Human mesenchymal stem cells were isolated by iliac crest aspiration andgrown in cell culture for 6 months (passage 19) prior to observing asignificant decrease in cell growth. Compounds of the present inventionwere added directly to the culture medium daily for 3 days. Significantsurvival and proliferation of the hMSC was observed upon treatment withvarious compounds of the present invention (FIG. 4).

The possibility that compounds of the present invention enhance hMSCsurvival and proliferation by activation of TERT was examined byculturing hMSC (p 3) in a tissue culture chamber slide at 17×10³cells/well for 60 h. Compounds of the present invention were added for 6h with fresh medium. Immunofluorescence with anti-hTERT antibody (firstantibody) and cy3 fluorescent secondary antibody was carried out. Thenucleus was stained with DAPI. Compounds of the present inventionactivated telomerase expression in hMSC (FIG. 5). Expression oftelomerase protein was seen in both cytoplasm and nucleus. Degree ofactivation of telomerase expression was Compound 68>79>77.Quantification of telomerase activity in hMSC was performed by Real timePCR (Quantification telomerase detection kit) in nuclear extractsderived from hMSC treated with compound 79 (250 nM) for 6 or 24 h.Compound 79 enhanced telomerase activity in hMSC by 12- or 6-fold in atime-dependent manner (FIG. 5B).

Example 14 Effects of Compounds of the Present Invention on HumanKeratinocytes

Human keratinocytes which had lost their ability to proliferate (intheir fifth passage) were obtained from a local skin bank and treatedwith Compound 68 for 24 h. Cell viability, cell number and morphologywas examined. Cells treated with compounds of the present inventionexhibited significant (2-4-fold) cell proliferation and viable cellswere observed (FIG. 6). Compound 79 exhibited a better cell survivaleffect than Compound 68. These results support a role for the compoundsof the present invention in skin transplantation, wound healing, chronicskin ulcers and other skin conditions.

Example 15 Effects of Compounds of the Present Invention on HumanRetinal Pigmented Epithelial Cells

Chronic exposure of human retinal pigmented epithelial (RPE) cells tooxidative stress predisposes them to the development of age-relatedmacular degeneration (AMD). Human RPE cells were exposed to oxidativestress by treatment with H₂O₂ (1 mM) for 24 hours, and H₂O₂ (0.5 mM) foranother 24 hours. The cells were then treated daily for two days withthe indicated compounds in the absence of H₂O₂. Increased cell growthwas observed as a consequence of treatment with the compounds (FIG. 7A).Telomerase activity was enhanced in RPE cells treated with compounds 68and 79 by 30- and 50-fold, respectively (FIG. 7B). These results suggesta role for the compounds of the present invention in treating maculardegeneration and other retinal diseases.

Example 16 Extension of Survival in C. elegans

Survival of the C. elegans, a nematode that normally survives for 12-14days, represents an acceptable model for studying life extension.Nematodes were administered tri-phenyl telomerase-activating compoundsand their effect on the nematode lifespan extension was determined.Nematodes were grown with the activators at a concentration of 50micromolar, and mean, maximum and half-life were measured in comparisonwith untreated controls. Lifespan increased by 13-43% (Table 2), and themedian lifespan increased approximately 2-fold (from 12 days to 22 days,as shown in FIG. 8).

TABLE 2 Extension of survival of C. elegans by telomerase compoundsCompound no. Lifespan extension (%) 61 30 62 29 68 13 77 43 78 36 79 16

Example 17 Activation of Telomerase Expression by Compounds of thePresent Invention in Rat Endometrial Cells

Female rats in diestrus (the stage at which the epithelial layer in therat endometrium undergoes degradation) were injected subcutaneously with6 mg/kg of Compound 68, or with a vehicle. The rats were sacrificed 24 hlater and endometrial sections were prepared from the uterine horns oftreated and untreated rats and analyzed by histological procedures andby immunohistochemical staining Endometrial slices were stained byhematoxylin-eosin. Treatment with compounds of the present inventionprevented the degradation of the endometrial epithelial layer. Inaddition, morphologically, the endometrial tissue resembled thestructure of the proliferating stages (proestrus and estrus) (FIG.9A,B). Activation of telomerase expression in the endometrial cells oftreated rats was examined by immunohistochemistry with anti-telomeraseantibody (FIG. 9C-F). There was a significant increase in the stainingof telomerase in the endometrial tissue of rat injected with compoundsof the present invention, especially in the epithelial layers of theendometrial lumen and the epithelial layer of the secreting glands(compare FIGS. 9C and D, E and F). In addition, telomerase activity wasmeasured in nuclear extracts derived from the endometrium of the varioustreatment groups. A significant 4-8-fold increased in the activity oftelomerase was observed in the rats injected with compounds of thepresent invention (FIG. 9G), indicating that compounds of the presentinvention injected subcutaneously in the neck reached the endometrialtissue, activated telomerase and caused tissue proliferation. These datasupport a role for the compounds of this invention activating telomerasein animals and affecting the proliferation of a tissue in vivo. In someembodiments of the invention, the data support a role for the treatmentof endometriosis with the compounds of this invention.

Example 18 Telomerase Activation of Rat Brain Cortex Cells by Compoundsof the Present Invention

The effect of compounds of the present invention injected subcutaneouslyinto female rats and adult male mice on telomerase expression in variousregions of the brain was examined. Co-localization of telomerase andDAPI (DAPI or 4′,6-diamidino-2-phenylindole), a DNA-binding fluorescentstain demonstrated activation and/or expression in these cells as aconsequence of treatment. A very low expression of telomerase wasobserved in specific cells in various areas of the CNS (FIGS. 10 and11). Injections of Compounds 79 and 77 into rat significantly activatedthe expression of telomerase in the brain cortex, but Compound 68 didnot, indicating that Compounds 79 and 77, but not Compound 68, may crossthe blood-brain barrier (BBB) (FIG. 10).

Compound 79 was injected subcutaneously into three-month-old male mice.The mice were sacrificed 24 h later and the cortex, cerebellum,hippocampus, hypothalamus, brain stem and olfactory bulb isolated andsubjected to immunofluorescence. A low expression of telomerase wasobserved in most of the brain regions examined except for Purkinje cellsin the cerebellum (FIG. 11). Injection of compounds of the presentinvention significantly and dramatically increased telomerase expressionin the various brain regions. This activation appeared to be specificfor certain neuronal cells. Compound 79, for example, increasedtelomerase expression in the motor-neuronal cells present in the brainstem.

Confirmation of immunofluorescence results was obtained by examining thelevel of TERT protein enzyme in nuclear protein extracts derived frombrains removed from rats treated or untreated with compounds of thepresent invention. Western blot analysis using specific anti-hTERTantibody was performed and telomerase activation (%) was determined andcalculated relative to the results obtained with the vehicle. Asignificant increase (9-13-fold) in telomerase protein was observed inrat brain cortex cells treated with compounds of the present invention(FIG. 12), supporting a role for the use of compounds of the presentinvention in treating brain-related diseases.

Example 19 Prevention of Glutamate-Induced Apoptosis in Mouse Cerebellum

The ability of compounds of the present invention to preventglutamate-induced apoptosis in mouse cerebellum was examined. Mice wereinjected with 6 mg/kg of Compound 79 or with vehicle only. Mice weresacrificed 24 h after treatment and their brains were removed.Cerebellum slices were prepared and subjected to glutamate treatment atvarious concentrations for 30 min. Sections were stained with propidiumiodide and the number of apoptotic cells was counted and calculatedusing specific software (Jimage). Prevention of apoptosis in micetreated with compounds of the present invention was calculated as apercentage relative to control mice (FIG. 13). This supports a role forcompounds of the present invention in treating seizures, stroke,Alzheimer's disease, epilepsy, schizophrenia, and alcohol and opiateaddition.

Example 20 Telomerase Activation of Mouse Heart Cells by Compounds ofthe Present Invention

The activation of telomerase by compounds of the present invention inheart protein extracts derived from one mouse was measured (FIG. 14).There was a significant increase in the heart telomerase expressionfollowing injection of Compound 79. This supports a role for compoundsof the present invention in treating infarct, ischemia, myocarditis,etc.

Example 21 Prevention of Drug Damage to Embryos by Compounds of thePresent Invention

The ability of compounds of the present invention to prevent thedamaging effects of drugs on embryonic development was examined in rats.Female rats were treated with camptothecin (CPT) (5 mg/kg), ananti-cancer drug, and then injected with Compound 68 (6 mg/kg) orvehicle (0.1%). Embryos were removed at days 14-15 of pregnancy andexamined for damage. Embryos treated with both CPT and Compound 68developed normally, while embryos treated with CPT only showed evidenceof abnormal development (FIG. 15).

Example 22 Effect of Compounds of the Present Invention on BCL1Tumorigenicity

(BALB/c×C57BL/6)F1 mice were inoculated with 10⁵ murine B-cell leukemialymphoma (BCL1) cells. Splenomegaly and lymphocytosis (leukemia)developed in all controls and in mice treated with telomerase-activatingcompounds (40 or 80 mg/kg).

TABLE 3 Experimental Group Survival Median (range) BCL1 only 29 (21-35)BCL1 + Compound 77 10 mM 26 (22-41)

Telomerase-activating compounds did not enhance the development ofleukemia and did not shorten the survival of mice which were inoculatedwith leukemia. The telomerase-activating compounds of the presentinvention did not facilitate progression of existing cancer, nor didlong-term administration of the compounds result in development ofcancer.

Example 23 Effect of Compounds of the Present Invention onGraft-Versus-Host Disease (GVHD) as a Representative T Cell Function

(BALB/c×C57BL/6)F1 mice were inoculated with 30×10⁶C57BL/6 spleen cellsfor induction of GVHD. Mice treated with Compound 77 exhibited moresevere signs of acute GVHD, as demonstrated by weight loss as comparedwith untreated controls. These results suggest that compounds of thepresent invention may be used as a powerful adjuvant.

Example 24 Effect of Compounds of the Present Invention onGraft-Versus-Leukemia Disease (GVL) as a Representative Example ofImmunotherapy by T Cells

(BALB/c×C57BL/6)F1 mice were inoculated with 10⁵ BCL1 following totalbody irradiation 400 cGy to allow engraftment of allogeneic lymphocytesfor induction of graft-versus-leukemia (GVL) effects medicated by30×10⁶C57BL/6 spleen cells. Survival of untreated controls ranged from21-35 days (median 29). Survival of mice treated with allogeneic stemcells ranged from 13-22 days since the leukemia activated a severe GVHD.In contrast, survival of mice treated with allogeneic stem cells andcompounds of the present invention ranged from 37-74 days, with onemouse surviving 125 days. This supports a role for the compounds of thepresent invention in treating leukemia.

While certain features of the invention have been illustrated anddescribed herein, many modifications, substitutions, changes, andequivalents will now occur to those of ordinary skill in the art. It is,therefore, to be understood that the appended claims are intended tocover all such modifications and changes as fall within the true spiritof the invention.

What is claimed is:
 1. A method of stimulating or increasing telomeraseactivity in an islet cell or in pancreatic tissue of a subject, saidmethod comprising contacting said islet cell or pancreatic tissue ofsaid subject with an effective amount of a compound represented by thestructure of formula I:

wherein Z is carbon, nitrogen, phosphor, arsenic, silicon or germanium;R₁ to R₉ are the same or different, H, D, OH, halogen, nitro, CN,nitrileamido, amidosulfide, amino, aldehyde, substituted ketone, —COOH,ester, trifluoromethyl, amide, substituted or unsubstituted alkyl,alkenyl, alkynyl, aryl, arylalkyl, alkylaryl, arylsulfonyl,arylalkylenesulfonyl, alkoxy, alkylalkoxy, haloalkyl, alkylhaloalkyl,haloaryl, aryloxy, amino, monoalkylamino, dialkylamino, alkylamido,arylamino, arylamido, alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or R₃, R₄, or R₇, forms a fusedcycloalkyl, heterocycloalkyl, aromatic or heteroaromatic ring with themain aromatic ring; and R₁₀ is nothing, H, D, OH, methyl, halogen, oxo,nitro, CN, nitrileamido, amidosulfide, amino, aldehyde, substitutedketone, —COOH, ester, trifluoromethyl, amide, substituted orunsubstituted alkyl, alkenyl, alkynyl, aryl, arylalkyl, alkylaryl,arylsulfonyl, arylalkylenesulfonyl, alkoxy, haloalkyl, haloaryl,cycloalkyl, alkylcycloalkyl, aryloxy, monoalkylamino, dialkylamino,alkylamido, arylamino, arylamido, alkylthio, arylthio, heterocycloalkyl,alkylheterocycloalkyl, heterocycloalkylalkyl, heteroaryl,hetroarylalkyl, alkylheteroaryl; or its isomer, pharmaceuticallyacceptable salt, pharmaceutical product, hydrate, N-oxide, polymorph,crystal or any combination thereof.
 2. The method of claim 1, whereinsaid compound is represented by the structure of formula IV:


3. The method of claim 2, wherein said compound is represented by thestructure of formula VI:

wherein R₁′, R₃′, R₄′, R₆′ R₇′, and R₉′ are the same or differentcomprising halogen, aryl, alkyl, cycloalkyl, heterocycloalkyl, alkoxy,monoalkylamino, dialkylamino or arylamino; and R₁₀ is as describedabove.
 4. The method of claim 3, wherein said compound is represented bythe structure of formula VII:


5. The method of claim 3, wherein said compound is represented by thestructure of formula VIII:


6. The method of claim 3, wherein said compound is represented by thestructure of formula IX:


7. The method of claim 3, wherein said compound is represented by thestructure of formula X:


8. The method of claim 3, wherein said compound is represented by thestructure of formula XI:


9. The method of claim 3, wherein said compound is represented by thestructure of formula XII:


10. The method of claim 3, wherein said compound is represented by thestructure of formula XIII:


11. The method of claim 1, wherein said compound is represented by thestructure of formula XIV:


12. The method of claim 1, wherein said compound is represented by thestructure of formula XV:


13. The method of claim 1, wherein said compound is represented by thestructure of formula XVI:


14. The method of claim 1, wherein said islet cell or pancreatic tissueis contacted with a pharmaceutical composition comprising said compoundand a pharmaceutically acceptable carrier.
 15. The method of claim 1,wherein said subject has diabetes.
 16. The method of claim 1, whereinsaid subject is predisposed to diabetes.
 17. The method of claim 1,wherein Z is carbon, silicon or germanium and R₁₀ is methyl.